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.

Immature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla migration of positively selected thymocytes in the thymus. However, the role of the cortex-to-medulla migration in T cell development and selection has remained unclear. The present study shows that the developmental kinetics and the thymic export of mature thymocytes were undisturbed in adult mice lacking CCR7 or its ligands (CCR7L). The inhibition of sphingosine-1-phosphate-mediated lymphocyte egress from the thymus led to the accumulation of mature thymocytes in the cortex of CCR7- or CCR7L-deficient mice, unlike the accumulation in the medulla of normal mice, thereby suggesting that mature thymocytes may be exported directly from the cortex in the absence of CCR7 signals. However, the thymocytes that were generated in the absence of CCR7 or CCR7L were potent in causing autoimmune dacryoadenitis and sialadenitis in mice and were thus incapable of establishing central tolerance to organ-specific antigens. These results indicate that CCR7-mediated cortex-to-medulla migration of thymocytes is essential for establishing central tolerance rather than for supporting the maturation or export of thymocytes.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-κB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.

Most T lymphocytes are generated within the thymus. It is unclear, however, how newly generated T cells relocate out of the thymus to the circulation. The present study shows that a CC chemokine CCL19 attracts mature T cells out of the fetal thymus organ culture. Another CC chemokine CCL21, which shares CCR7 with CCL19 but has a unique C-terminal extension containing positively charged amino acids, failed to show involvement in thymic emigration. Neonatal appearance of circulating T cells was defective in CCL19-neutralized mice as well as in CCR7-deficient mice but not in CCL21-neutralized mice. In the thymus, CCL19 is predominantly localized in the medulla including endothelial venules. These results indicate a CCL19- and CCR7-dependent pathway of thymic emigration, which represents a major pathway of neonatal T cell export.

The systemic administration of keratinocyte growth factor (KGF) enhances T-cell lymphopoiesis in normal mice and mice that received a bone marrow transplant. KGF exerts protection to thymic stromal cells from cytoablative conditioning and graft-versus-host disease-induced injury. However, little is known regarding KGF's molecular and cellular mechanisms of action on thymic stromal cells. IntroductionDecreased T-cell cellularity and a skewed TCR repertoire are hallmarks of an immune deficiency commonly observed in old age, as a consequence of general infectious diseases and aggressive lymphocyte-depleting therapies for diverse malignancies. [1][2][3][4] The regeneration of a phenotypically and functionally normal T-cell compartment is curtailed for an extended period of time in patients receiving a hematopoietic stem cell transplant (HSCT). [5][6][7] This lack in T-cell reconstitution is associated with opportunistic infections, the reactivation of latent viral and parasitic infections, chronic inflammation, and autoimmunity. 3,4 Following cytoablative therapy, the recovery of the T-cell compartment relies on 2 independent pathways, that is, the expansion of peripheral T cells and, alternatively, the de novo production of T cells in the thymus. 1,2,7-10 The latter assures the generation of a population of naive T cells expressing a diverse repertoire of TCR specificities. 5,7,8,10,11 The extent of thymusdependent T-cell reconstitution correlates directly with thymic size following immune ablation and hematopoietic stem cell (HSC)-derived reconstitution 7,12 but is inversely related to age and transplant-related toxicities such as graft-versus-host disease (GVHD). 10,[13][14][15][16][17] The generation of new T cells of donor origin depends on the migration of hematopoietic precursors to the thymus. Normal thymic T-cell development is in turn contingent on the regular maintenance of the stromal microenvironment. However, age-related thymic involution 18 and injury from radiation, 19 GVHD, 20 chemotherapy, 12,21 or infection 3,4,12,[18][19][20][21][22][23] preclude normal thymopoiesis to occur as they directly affect thymic epithelial cells (TECs). There has been considerable interest in identifying strategies to prevent TEC injury. Recently, robust T-cell lymphopoiesis has been maintained in myeloablated HSCT recipients by pretransplantation administration of different factors such as 24,25 androgen antagonists, 26 and fibroblast growth factor 7 (Fgf7; aka, keratinocyte growth factor [KGF]). 20,27-29 KGF belongs to the family of the structurally related Fgfs and is a potent epithelial cell mitogen. 27,30 KGF is expressed under physiological conditions within the thymus both by mesenchymal cells and by T cells at specific developmental stages. To exert its biologic activity, KGF activates the IIIb variant of the FgfR2 receptor (FgfR2IIIb), which is expressed within the thymus exclusively on TECs. 31 Experiments using mice deficient for FgfR2IIIb or the removal of mesenchyme from normal embryos revealed the importa...

During embryonic development, T-lymphoid precursor cells colonize the thymus. Chemoattraction by the fetal thymus is thought to mediate T-precursor cell colonization. However, the molecules that attract Tprecursor cells to the thymus remain unclear. By devising time-lapse visualization in culture, the present results show that alymphoid fetal thymus lobes attract Tprecursor cells from fetal liver or fetal blood. CD4 ؊ CD8 ؊ CD25 ؊ CD44 ؉ fetal thymocytes retained the activity to specifically re-enter the thymus. The attraction was predominantly due to I-A-expressing thymic epithelial cells and was mediated by pertussis toxin-sensitive G-protein signals. Among the chemokines produced by the fetal thymus, CCL21, CCL25, and CXCL12 could attract CD4 ؊ CD8 ؊ CD25 ؊ -CD44 ؉ fetal thymocytes. However, fetal thymus colonization was markedly diminished by neutralizing antibodies specific for CCL21 and CCL25, but not affected by anti-CXCL12 antibody. Fetal thymus colonization was partially defective in CCL21-deficient plt/plt mice and was further diminished by anti-CCL25 antibody. These results indicate that CCL21 is involved in the recruitment of T-cell precursors to the fetal thymus and suggest that the combination of CCL21 and CCL25 plays a major role in fetal thymus colonization. (Blood. 2005;105:31-39)