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SummaryThe thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire+ mTECs at this key developmental stage, prior to αβ T cell repertoire selection, was jointly directed by Rankl+ lymphoid tissue inducer cells and invariant Vγ5+ dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire+ mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5+ γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire+ mTEC maturation.

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire+ mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall’s corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire+ mTEC development, use of an inducible ZAP70 transgenic mouse line—in which positive selection can be temporally controlled—demonstrates that the emergence of involucrin+ mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire+ mTECs depends upon RANK signaling, continued mTEC development to the involucrin+ stage maps to activation of the LTα–LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

In the thymus, interactions with both cortical and medullary microenvironments regulate the development of self-tolerant conventional CD4+ and CD8+ αβT cells expressing a wide range of αβTCR specificities. Additionally, the cortex is also required for the development of invariant NKT (iNKT) cells, a specialized subset of T cells that expresses a restricted αβTCR repertoire and is linked to the regulation of innate and adaptive immune responses. Although the role of the cortex in this process is to enable recognition of CD1d molecules expressed by CD4+CD8+ thymocyte precursors, the requirements for additional thymus microenvironments during iNKT cell development are unknown. In this study, we reveal a role for medullary thymic epithelial cells (mTECs) during iNKT cell development in the mouse thymus. This requirement for mTECs correlates with their expression of genes required for IL-15 trans-presentation, and we show that soluble IL-15/IL-15Rα complexes restore iNKT cell development in the absence of mTECs. Furthermore, mTEC development is abnormal in iNKT cell–deficient mice, and early stages in iNKT cell development trigger receptor activator for NF-κB ligand–mediated mTEC development. Collectively, our findings demonstrate that intrathymic iNKT cell development requires stepwise interactions with both the cortex and the medulla, emphasizing the importance of thymus compartmentalization in the generation of both diverse and invariant αβT cells. Moreover, the identification of a novel requirement for iNKT cells in thymus medulla development further highlights the role of both innate and adaptive immune cells in thymus medulla formation.

SUMMARY Lymph node development during embryogenesis involves lymphotoxin-β receptor engagement and subsequent differentiation of a poorly defined population of mesenchymal cells into lymphoid tissue organizer cells. Here, we showed that embryonic mesenchymal cells with characteristics of adipocyte precursors present in the microenvironment of lymph nodes gave rise to lymph node organizer cells. Signaling through the lymphotoxin-β receptor controlled the fate of adipocyte precursor cells by blocking adipogenesis and instead promoting lymphoid tissue stromal cell differentiation. This effect involved activation of the NF-κB2-RelB signaling pathway and inhibition of the expression of the key adipogenic factors Pparγ and Cebpα. In vivo organogenesis assays show that embryonic and adult adipocyte precursor cells can migrate into newborn lymph nodes and differentiate into a variety of lymph node stromal cells. Thus, we propose that adipose tissues act as a source of lymphoid stroma for lymph nodes and other lymphoid structures associated with fat.

The thymus supports the production of self-tolerant T cells from immature precursors. Studying the mechanisms regulating the establishment and maintenance of stromal microenvironments within the thymus therefore is essential to our understanding of T-cell production and ultimately immune system functioning. Despite our ability to phenotypically define stromal cell compartments of the thymus, the mechanisms regulating their development and the ways by which they influence T-cell precursors are still unclear. Here, we review recent findings and highlight unresolved issues relating to the development and functioning of thymic stromal cells.

Lymphocytes from lymphotoxin (LT)␣ IntroductionThe development of segregated B-cell and T-cell areas within secondary lymphoid organs is the platform for the development of both high-affinity class-switched antibodies and memory antibody responses; neither of these functions develops in lymphotoxin (LT) ␣ Ϫ/Ϫ mice, in which there is no B/T segregation. 1 The absence of segregation is due to impaired organization rather than intrinsic defects in the lymphocytes themselves, as LT␣ Ϫ/Ϫ lymphocytes both segregate and function normally following transfer into irradiated normal 2 or RAG-deficient 1 hosts, which lack B and T cells. A cellular source other than mature B or T cells is therefore implicated in the process of organization.LT␤R signals and perhaps TNFR1 signals mediate lymphoid B/T segregation by activating subpopulations of stromal cells, which then switch on the expression of chemokine genes. 3 The expression of CCR7 ligand attracts dendritic cells (DCs) and T cells to form the T-cell area 4 ; the expression of CXCR5 ligand brings B cells together to form follicles. 5 The genes for these receptors, TNFR1 and LT␤R, are tightly linked on chromosome 12 in humans and chromosome 6 in mice, implying that they arose by local gene duplication prior to speciation of human and mouse.The expression of the T-zone chemokines in lymph nodes is normal in RAG Ϫ/Ϫ mice, although the expression of the B-zone chemokine, CXCL13, is reduced to approximately 20%, and normal expression depends on B cells. 6 Along with the LT␣ Ϫ/Ϫ lymphocyte transfer experiments, these data suggest that there is a non-B non-T cell capable of inducing normal (T zone) and partial (B zone) chemokine expression in stroma.In this paper, we extend previous observations demonstrating a role for a non-B non-T cell in B/T segregation, 2 and identify CD4 ϩ CD3 Ϫ cells that we have previously implicated in T-cell memory for antibody responses in adult mice 7,8 as playing a role in the lymphoid stromal organization within secondary lymphoid tissues. We report that adult CD4 ϩ CD3 Ϫ cells express high levels of mRNA for LT␣, LT␤, tumor necrosis factor (TNF) ␣, and LIGHT, which are the ligands for TNFR1 and the LT␤R. Levels of expression are comparable with those expressed in embryonic and neonatal CD4 ϩ CD3 Ϫ cells, and the expression of LT␤ is at least an order of magnitude greater than in CD11c ϩ DCs or plasmacytoid DCs (pDCs). Furthermore, using adoptive cell-transfer experiments, we demonstrate that the expression of these genes is functional: fetal CD4 ϩ CD3 Ϫ cells derived from embryonic day (E) 15 spleen and adult CD4 ϩ CD3 Ϫ cells, but not lymphocytes, pDCs, and DCs, are able to restore a significant degree of B/T segregation in the spleens of LT␣ Ϫ/Ϫ mice, and up-regulate VCAM-1 and CCL21 protein expression on the stroma.Using confocal microscopy, we demonstrate that this CD4 ϩ CD3 Ϫ cell associates closely with VCAM-1 ϩ follicular dendritic cells (FDCs) in B-cell areas as well as with the VCAM-1 ϩ stromal population within the T zone. Materials and ...

The thymus medulla prevents T cell–driven autoimmunity via central tolerance. Cosway et al. show that this specialization occurs independently of the topology that classically defines its structure and demonstrate that medulla function requires LTβR-mediated regulation of dendritic cells for negative selection.