Phenotypic and functional analysis of the mesenchymal stromal cell compartment in the thymus

Abstract: T cells are key cellular components of the adaptive immune system that play a critical role in our defense against pathogens and cancer but also contribute to pathology in the setting of chronic inflammation and autoimmunity. The thymus constitutes the body's major T cell production site, where bone marrow-derived progenitors migrate in a co-ordinated fashion through anatomically distinct cortical and medullary regions that are composed of epithelial and mesenchymal stromal elements. The interaction of cortical (cTEC) and medullary (mTEC) thymic epithelial cells (TEC) with migrating thymocytes is of fundamental importance in driving thymocyte differentiation and their selection towards generation of an immunocompetent but self-tolerant T cell repertoire. While the ontogeny, phenotype and functionality of TEC subsets has been extensively studied, far less is known regarding the thymic mesenchymal cell (TMC) compartment. The vitamin A metabolite, retinoic acid (RA) is a potent regulator of cell proliferation, differentiation and apoptosis that impacts on gene transcription by acting as a trans-activating ligand for a family of transcription factors called RA receptors (RARs). RA-dependent RAR-mediated events (also called RA signaling) have been widely implicated in organ development and epithelial tissue homeostasis but the putative role of endogenous RA signals in regulating TEC and/or thymocyte homeostasis has remained unknown. In this thesis, using comprehensive phenotypic, genetic lineage tracing and microarray analyses we demonstrate that postnatal TMCs comprise two phenotypically distinct and functionally specialized neural crest-derived, gp38+ and gp38- TMC subsets. We characterize gp38- TMC as mural cells with classical pericyte and vascular smooth muscle cell functions that intimately associate with and support thymic vascular endothelium. In contrast, gp38+ TMC were central producers of a range of factors implicated in regulating TEC homeostasis. Our data also provide an initial description of the neural crest cell maturation pathway towards mature gp38+ and gp38- TMC during thymic ontogeny. Using gene expression, biochemical and functional analyses, we further identify gp38+ TMCs together with a subset of mTECs as the major cell populations capable of RA production in the post-natal thymus. In functional experiments involving FTOC, we show that mesenchymal cell-derived RA plays an important role in regulating cTEC numbers and provide initial evidence for a direct role of RA responses in TEC in regulating cTEC homeostasis in vivo. In addition to its direct role in regulating TEC compartments, we demonstrate the presence of endogenous RA sensing by developing thymocytes, and further provide initial data suggesting a key and direct role for RA in thymocyte development in vivo. Collectively, our results significantly broaden the current understanding of TMC heterogeneity, ontogeny and function, and provide valuable insights to the current understanding of vitamin A metabolism and the functionality of RA in the thymus.