Immunosenescence caused by age-associated involution of the thymus poses a significant risk to the aging
human population. During age-associated thymic involution, a reduction and disorganization of the functional
regions of the thymus, along with an increase in adipogenic and other undesirous cell types, results in a lower
capacity of the thymus to generate functional T-cells required for adaptive immunity. Currently, the cellular
origins of thymic adipocytes remain obscure, with some investigators suggesting that adipocytes infiltrate the
thymus during involution and others indicating that they differentiate directly from thymic stromal cells. Our lab
has identified a potential regulator of adipogenesis in the thymus, the Activating Transcription Factor 3 (ATF3),
in which deletion of the Atf3 gene results in an increased presence of lipid-laden thymic stromal cells at 2-
months of age in mice when assessed by both flow cytometry and immunohistochemistry. At 10-months of
age, FACs analysis of adipogenic cells in Atf3 mutants and wild-type controls show that they are similar
quantitatively. However, qualitative analysis by immunohistochemistry reveals differences in adipogenic cell
types in Atf3 homozygous and heterozygous mutants, which warrants further investigation. We have also
conducted a lineage trace study using Atf3null/null; Foxn1Cre/+; Rosa26Tom/+ mice to identify if a subset of thymic
epithelial cells (TECs) undergo adipogenesis by using imaging flow cytometry. We identified four major
classes of TEC-derived adipogenic cells (LipidTox+EpCAM+Ly51+, LipidTox+EpCAM+Ly51-, LipidTox+EpCAM-
Ly51-, and LipidTox+EpCAM-Ly51+ cells), which include lipid-laden cTECs and mTECs. In frozen thymic
sections, we have identified lipid-laden cTECs that express PPARy starting at 6, 7, and 10-months of age. In
addition, we identified adipogenic mTECs that express FSP1, a marker for EMT, when we looked at 10-month
old mice. These findings suggest that although both cTECs and mTECs become adipogenic, each may be
regulated by different molecular mechanisms. We have also looked at tissue from a PPAR¿-tdTomato reporter
mouse engineered by the laboratory of Diane Mathis at Harvard. Using the reporter mice, we have identified
adipogenic vascular-associated cells and the presence of globular fat cells within the lumen of the vasculature
that appear to be infiltrating the thymus. Overall, we have begun to characterize the different classes of thymic
adipocytes more fully, and we are beginning to understand more about the genes that may be governing the
process. We have optimized techniques to study thymic adipocytes using standard and imaging flow cytometry
and immunocytochemistry. We have access to a novel mouse line, the PPARy-tdTomato line, that we plan on
using for our research purposes. These data suggest that both cTECs and mTECs give rise to a subpopulation
of thymic adipocytes and that ATF3 is a likely repressor of adipogenesis during thymic involution. In Aim 1, we
will identify the cell type in which ATF3 acts to regulate thymic adipogenesis. In Aim 2, we will identify the
mechanisms by which ATF3 regulates adipogenesis.