Despite the importance of thymic stromal cells to T-cell development, relatively little is known about their biology. Here, we use single-cell analysis of stromal cells to analyze extensive changes in the number and composition of thymic stroma throughout life, revealing a surprisingly dynamic population. Phenotypic progression of thymic epithelial subsets was assessed at high resolution in young mice to provide a developmental framework. The cellular and molecular requirements of adult epithelium were studied, using various mutant mice to demonstrate new cross talk checkpoints dependent on RelB in the cortex and CD40 in the medulla. With the use of Ki67 and BrdU labeling, the turnover of thymic epithelium was found to be rapid, but then diminished on thymic involution. The various defects in stromal turnover and composition that accompanied involution were rapidly reversed following sex steroid ablation. Unexpectedly, mature cortical and medullary epithelium showed a potent capacity to stimulate naive T cells, comparable to that of thymic dendritic cells. Overall, these studies show that the thymic stroma is a surprisingly dynamic population and may have a more direct role in negative selection than previously thought. IntroductionT-cell development in the thymus is essential for the establishment and maintenance of the adaptive immune system. Thymic stromal cells mediate various phases of thymocyte development to produce mature T cells capable of responding to foreign antigen while remaining tolerant of self. It is well established that different subsets of stromal cells form microenvironments in the thymic subcapsule, cortex, and medulla to facilitate distinct thymocyte maturational steps. 1 The maintenance of thymic microenvironments requires reciprocal interactions between thymocytes and stromal cells, termed thymic cross talk. [2][3][4] Although much is known about the biology of thymocytes, our understanding of thymic stromal cells is lacking.The heterogeneity of murine thymic stromal cells has been demonstrated using monoclonal antibodies specific for various stromal determinants (eg, Godfrey et al 5 ). These have proven useful in defining the epithelium, endothelium, fibroblasts, dendritic cells (DCs), and macrophages within thymic microenvironments. The cortex is characterized by a meshwork of reticular, cortical thymic epithelial cells (cTECs) that can be identified by distinctive patterns of intracellular (eg, MTS-44, K8) and surface (eg, Ly51) antigen expression. [5][6][7] Early T-cell development involves the migration of immature double-negative (DN, CD4 Ϫ CD8 Ϫ ) thymocytes through the thymic cortex toward the subcapsule in close contact with cTECs. 8 At the subcapsule, lining fibroblasts facilitate DN thymocyte development through the provision of extracellular matrix components. 9 Maturing thymocytes then return through the cortex where cTECs mediate the process of positive selection, rescuing T-cell receptor positive (TCR ϩ ) double-positive (DP; CD4 ϩ CD8 ϩ ) thymocytes capable of interactin...

Upon TCR-mediated positive selection, developing thymocytes relocate within the thymus from the cortex to the medulla for further differentiation and selection. However, it is unknown how this cortex–medulla migration of thymocytes is controlled and how it controls T cell development. Here we show that in mice deficient for CCR7 or its ligands mature single-positive thymocytes are arrested in the cortex and do not accumulate in the medulla. These mutant mice are defective in forming the medullary region of the thymus. Thymic export of T cells in these mice is compromised during the neonatal period but not in adulthood. Thymocytes in these mice show no defects in maturation, survival, and negative selection to ubiquitous antigens. TCR engagement of immature cortical thymocytes elevates the cell surface expression of CCR7. These results indicate that CCR7 signals are essential for the migration of positively selected thymocytes from the cortex to the medulla. CCR7-dependent cortex–medulla migration of thymocytes plays a crucial role in medulla formation and neonatal T cell export but is not essential for maturation, survival, negative selection, and adult export of thymocytes.

Autoimmune regulator (AIRE) gene mutation is responsible for the development of organ-specific autoimmune disease with monogenic autosomal recessive inheritance. Although Aire has been considered to regulate the elimination of autoreactive T cells through transcriptional control of tissue-specific Ags in thymic epithelial cells, other mechanisms of AIRE-dependent tolerance remain to be investigated. We have established Aire-deficient mice and examined the mechanisms underlying the breakdown of self-tolerance. The production and/or function of immunoregulatory T cells were retained in the Aire-deficient mice. The mice developed Sjögren’s syndrome-like pathologic changes in the exocrine organs, and this was associated with autoimmunity against a ubiquitous protein, α-fodrin. Remarkably, transcriptional expression of α-fodrin was retained in the Aire-deficient thymus. These results suggest that Aire regulates the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus, at least against this ubiquitous protein. Rather, Aire may regulate the processing and/or presentation of self-proteins so that the maturing T cells can recognize the self-Ags in a form capable of efficiently triggering autoreactive T cells. With the use of inbred Aire-deficient mouse strains, we also demonstrate the presence of some additional factor(s) that determine the target-organ specificity of the autoimmune disease caused by Aire deficiency.