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αβT cells are an essential component of effective immune responses. The heterogeneity that lies within them includes subsets that express diverse self-MHC-restricted αβT cell receptors, which can be further subdivided into CD4+ helper, CD8+ cytotoxic, and Foxp3+ regulatory T cells. In addition, αβT cells also include invariant natural killer T cells that are very limited in αβT cell receptor repertoire diversity and recognise non-polymorphic CD1d molecules that present lipid antigens. Importantly, all αβT cell sublineages are dependent upon the thymus as a shared site of their development. Ongoing research has examined how the thymus balances the intrathymic production of multiple αβT cell subsets to ensure correct formation and functioning of the peripheral immune system. Experiments in both wild-type and genetically modified mice have been essential in revealing complex cellular and molecular mechanisms that regulate thymus function. In particular, studies have demonstrated the diverse and critical role that the thymus medulla plays in shaping the peripheral T cell pool. In this review, we summarise current knowledge on functional properties of the thymus medulla that enable the thymus to support the production of diverse αβT cell types.

Therapeutic interventions used for cancer treatment provoke thymus damage and limit the recovery of protective immunity. Here, we show that eosinophils are an essential part of an intrathymic type 2 immune network that enables thymus recovery after ablative therapy. Within hours of damage, the thymus undergoes CCR3-dependent colonization by peripheral eosinophils, which reestablishes the epithelial microenvironments that control thymopoiesis. Eosinophil regulation of thymus regeneration occurs via the concerted action of NKT cells that trigger CCL11 production via IL4 receptor signaling in thymic stroma, and ILC2 that represent an intrathymic source of IL5, a cytokine that therapeutically boosts thymus regeneration after damage. Collectively, our findings identify an intrathymic network composed of multiple innate immune cells that restores thymus function during reestablishment of the adaptive immune system.

The transcription factor FOXN1 is a master regulator of thymic epithelial cell (TEC) development and function. Here, we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multimolecular nuclear condensates essential for the factor’s transcriptional activity. FOXN1’s C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are altered in a patient with a mutant FOXN1 that is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog selectively impairs mouse TEC differentiation, revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect., Science Advances, 7 (49), ISSN:2375-2548

The thymus ensures immune tolerance by synchronizing thymocyte development and selection. Alawam et al. show that a functional uncoupling of these events occurs after bone marrow transplantation, which results in post-transplant tolerance loss and autoimmunity caused by selective failures in thymus medulla regeneration., Bone marrow transplantation (BMT) is a widely used therapy for blood cancers and primary immunodeficiency. Following transplant, the thymus plays a key role in immune reconstitution by generating a naive αβT cell pool from transplant-derived progenitors. While donor-derived thymopoiesis during the early post-transplant period is well studied, the ability of the thymus to synchronize T cell development with essential tolerance mechanisms is poorly understood. Using a syngeneic mouse transplant model, we analyzed T cell recovery alongside the regeneration and function of intrathymic microenvironments. We report a specific and prolonged failure in the post-transplant recovery of medullary thymic epithelial cells (mTECs). This manifests as loss of medulla-dependent tolerance mechanisms, including failures in Foxp3+ regulatory T cell development and formation of the intrathymic dendritic cell pool. In addition, defective negative selection enables escape of self-reactive conventional αβT cells that promote autoimmunity. Collectively, we show that post-transplant T cell recovery involves an uncoupling of thymopoiesis from thymic tolerance, which results in autoimmune reconstitution caused by failures in thymic medulla regeneration., Graphical Abstract

The release of newly selected αβT cells from the thymus is key in establishing a functional adaptive immune system. Emigration of the first cohorts of αβT cells produced during the neonatal period is of particular importance, because it initiates formation of the peripheral αβT-cell pool and provides immune protection early in life. Despite this, the cellular and molecular mechanisms of thymus emigration are poorly understood. We examined the involvement of diverse stromal subsets and individual chemokine ligands in this process. First, we demonstrated functional dichotomy in the requirement for CCR7 ligands and identified CCL21, but not CCL19, as an important regulator of neonatal thymus emigration. To explain this ligand-specific requirement, we examined sites of CCL21 production and action and found Ccl21 gene expression and CCL21 protein distribution occurred within anatomically distinct thymic areas. Although Ccl21 transcription was limited to subsets of medullary epithelium, CCL21 protein was captured by mesenchymal stroma consisting of integrin α7+ pericytes and CD34+ adventitial cells at sites of thymic exit. This chemokine compartmentalization involved the heparan sulfate–dependent presentation of CCL21 via its C-terminal extension, explaining the absence of a requirement for CCL19, which lacks this domain and failed to be captured by thymic stroma. Collectively, we identified an important role for CCL21 in neonatal thymus emigration, revealing the importance of this chemokine in initial formation of the peripheral immune system. Moreover, we identified an intrathymic mechanism involving cell-specific production and presentation of CCL21, which demonstrated a functional synergy between thymic epithelial and mesenchymal cells for αβT-cell emigration.

Autoimmune regulator+ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire+mTECs differentiate further into Post‐Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate‐mapping mouse models. Herein, we resolve this limitation by segmenting the mTEChi(MHCIIhiCD80hi) compartment into mTECA/hi (CD24−Sca1−), mTECB/hi (CD24+Sca1−), and mTECC/hi (CD24+Sca1+). While mTECA/hi included mostly Aire‐expressing cells, mTECB/hi contained Aire+ and Aire− cells and mTECC/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor‐product relationship between these subsets. Strikingly, transcriptomic analysis of mTECA/hi, mTECB/hi, and mTECC/hi sequentially mirrored the specific genetic program of Early‐, Late‐ and Post‐Aire mTECs. Corroborating their Post‐Aire nature, mTECC/hi downregulated the expression of tissue‐restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire‐deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation., Although the expression of Aire was initially considered to mark a terminal stage of mTEC maturation, recent studies have demonstrated that mTECs extend their differentiation beyond Aire. Integrating flow cytometry, functional, and transcriptomic analyses, Ferreirinha et al. provide an effective phenotypic‐based method to resolve Early‐, Late‐ and Post‐Aire mTECs.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

WNK1, a kinase that controls kidney salt homeostasis, also regulates adhesion and migration in CD4+ T cells. Wnk1 is highly expressed in thymocytes, and since migration is important for thymocyte maturation, we investigated a role for WNK1 in mouse thymocyte development. We find that WNK1 is required for the transition of double negative (DN) thymocytes through the β-selection checkpoint and subsequent proliferation and differentiation into double positive (DP) thymocytes. Furthermore, we show that WNK1 negatively regulates LFA1-mediated adhesion and positively regulates CXCL12-induced migration in DN thymocytes. Despite this, migration defects of WNK1-deficient thymocytes do not account for the developmental arrest. Instead, we show that in DN thymocytes WNK1 transduces pre-TCR signals via OXSR1 and STK39 kinases, and the SLC12A2 ion co-transporter that are required for post-transcriptional upregulation of MYC and subsequent proliferation and differentiation into DP thymocytes. Thus, a pathway regulating ion homeostasis is a critical regulator of thymocyte development.

The thymus supports multiple αβ T cell lineages that are functionally distinct, but mechanisms that control this multifaceted development are poorly understood. Here we examine medullary thymic epithelial cell (mTEC) heterogeneity and its influence on CD1d-restricted iNKT cells. We find three distinct mTEClow subsets distinguished by surface, intracellular and secreted molecules, and identify LTβR as a cell-autonomous controller of their development. Importantly, this mTEC heterogeneity enables the thymus to differentially control iNKT sublineages possessing distinct effector properties. mTEC expression of LTβR is essential for the development thymic tuft cells which regulate NKT2 via IL-25, while LTβR controls CD104+CCL21+ mTEClow that are capable of IL-15-transpresentation for regulating NKT1 and NKT17. Finally, mTECs regulate both iNKT-mediated activation of thymic dendritic cells, and iNKT availability in extrathymic sites. In conclusion, mTEC specialization controls intrathymic iNKT cell development and function, and determines iNKT pool size in peripheral tissues., Thymus is a unique environment hosting the development of many T cell subsets with distinct functions. Here the authors show that medullary thymic epithelial cells (mTEC) are functionally diverse, with LTβR signaling serving differential regulation of mTEC for specific control of multiple lineages of invariant natural killer T cells.

Most αβ T cells that form in the thymus are generated during mainstream conventional thymocyte development and involve the generation and selection of a diverse αβ TCR repertoire that recognizes self-peptide/MHC complexes. Additionally, the thymus also supports the production of T cell subsets that express αβ TCRs but display unique developmental and functional features distinct from conventional αβ T cells. These include multiple lineages of CD1d-restricted invariant NKT (iNKT) cells that express an invariant αβ TCR, branch off from mainstream thymocytes at the CD4+CD8+ stage, and are potent producers of polarizing cytokines. Importantly, and despite their differences, iNKT cells and conventional αβ T cells share common requirements for thymic epithelial microenvironments during their development. Moreover, emerging evidence suggests that constitutive cytokine production by iNKT cells influences both conventional thymocyte development and the intrathymic formation of additional innate CD8+ αβ T cells with memory-like properties. In this article, we review evidence for an intrathymic innate lymphocyte network in which iNKT cells play key roles in multiple aspects of thymus function.

Summary Primary stimulation of T cells is believed to trigger unidirectional differentiation from naive to effector and memory subsets. Here we demonstrate that IL-7 can drive the phenotypic reversion of recently differentiated human central and effector memory CD8+ T cells into a naive-like phenotype. These “naive-revertant” cells display a phenotype similar to that of previously reported stem cell memory populations and undergo rapid differentiation and functional response following secondary challenge. The chromatin landscape of reverted cells undergoes substantial epigenetic reorganization with increased accessibility for cytokine-induced mediators such as STAT and closure of BATF-dependent sites that drive terminal differentiation. Phenotypic reversion may at least partly explain the generation of “stem cell memory” CD8+ T cells and reveals cells within the phenotypically naive CD8+ T cell pool that are epigenetically primed for secondary stimulation. This information provides insight into mechanisms that support maintenance of T cell memory and may guide therapeutic manipulation of T cell differentiation., Graphical Abstract, Highlights • γ-chain cytokines revert newly differentiated CD8+ T cells to a naive-like phenotype • These “naive-revertant” are primed for secondary challenge • Their chromatin landscape is reminiscent of memory cells • Specific signaling pathways and transcription factors are involved, Biological Sciences; Molecular Biology; Immunology

Despite the key role of primary and secondary lymphoid organ stroma in immunity, our understanding of the heterogeneity and ontogeny of these cells remains limited. Here, we identify a functionally distinct subset of BP3(-)PDPN(+)PDGFR beta(+)/alpha(+)CD34(+) stromal adventitial cells in both lymph nodes (LNs) and thymus that is located within the vascular niche surrounding PDPN(-)PDGFR beta(+)/alpha(-)Esam-1(+)ITGA7(+) pericytes. CD34(+) adventitial cells developed in late embryonic thymus and in postnatal LNs and in the thymus originated, along with pericytes, from a common anlage-seeding progenitor population. Using lymphoid organ re-aggregate grafts, we demonstrate that adult CD34(+) adventitial cells are capable of differentiating into multiple lymphoid stroma-like subsets including pericyte- ,FRC-, MRC-, and FDC-like cells, the development of which was lymphoid environment-dependent. These findings extend the current understanding of lymphoid mesenchymal cell heterogeneity and highlight a role of the CD34(+) adventitia as a potential ubiquitous source of lymphoid stromal precursors in postnatal tissues.

Thymic emigration is essential for establishing T cell immunity. We show the requirement for LTβR segregates from its control of medullary epithelium. Instead, our study demonstrates LTβR expression by the endothelium acts to rate limit thymocyte egress via perivascular routes., The emigration of mature thymocytes from the thymus is critical for establishing peripheral T cell compartments. However, the pathways controlling this process and the timing of egress in relation to postselection developmental stages are poorly defined. Here, we reexamine thymocyte egress and test current and opposing models in relation to the requirement for LTβR, a regulator of thymic microenvironments and thymocyte emigration. Using cell-specific gene targeting, we show that the requirement for LTβR in thymocyte egress is distinct from its control of thymic epithelium and instead maps to expression by endothelial cells. By separating emigration into sequential phases of perivascular space (PVS) entry and transendothelial migration, we reveal a developmentally ordered program of egress where LTβR operates to rate limit access to the PVS. Collectively, we show the process of thymic emigration ensures only the most mature thymocytes leave the thymus and demonstrate a role for LTβR in the initiation of thymus emigration that segregates from its control of medulla organization., Graphical Abstract

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal alpha beta T-cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC-II(low)CD40(-) cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin(+) thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1(-/-) mice. Moreover, CCRL1(-/-) mice have no major perturbations in T-cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4

White et al. describe a new mechanism of thymus emigration that is controlled by expression of the type 2 IL-4 receptor by thymic stroma and production of IL-4 and IL-13 by thymic-resident invariant NKT cells., In the thymus, stromal microenvironments support a developmental program that generates mature T cells ready for thymic exit. The cellular and molecular specialization within thymic stromal cells that enables their regulation of specific stages of thymocyte development is poorly understood. Here, we show the thymic microenvironment expresses the type 2 IL-4R complex and is functionally responsive to its known ligands, IL-4 and IL-13. Absence of IL-4Rα limits thymocyte emigration, leading to an intrathymic accumulation of mature thymocytes within medullary perivascular spaces and reduced numbers of recent thymic emigrants. Thymus transplantation shows this requirement maps to IL-4Rα expression by stromal cells, and we provide evidence that it regulates thymic exit via a process distinct from S1P-mediated migration. Finally, we reveal a cellular mechanism by which IL-4+IL-13+ invariant NKT cells are necessary for IL-4Rα signaling that regulates thymic exit. Collectively, we define a new axis for thymic emigration involving stimulation of the thymic microenvironment via type 2 cytokines from innate T cells.

Summary The thymus is a primary lymphoid tissue that supports the generation of αβT cells. In this review, we describe the processes that give rise to the thymus medulla, a site that nurtures self‐tolerant T‐cell generation following positive selection events that take place in the cortex. To summarize the developmental pathways that generate medullary thymic epithelial cells (mTEC) from their immature progenitors, we describe work on both the initial emergence of the medulla during embryogenesis, and the maintenance of the medulla during postnatal stages. We also investigate the varying roles that receptors belonging to the tumor necrosis factor receptor superfamily have on thymus medulla development and formation, and highlight the impact that T‐cell development has on thymus medulla formation. Finally, we examine the evidence that the thymic medulla plays an important role during the intrathymic generation of distinct αβT‐cell subtypes. Collectively, these studies provide new insight into the development and functional importance of medullary microenvironments during self‐tolerant T‐cell production in the thymus.

Phylogeny shows that CD4 T cell memory and lymph nodes coevolved in placental mammals. In ontogeny, retinoic acid orphan receptor (ROR)γ-dependent lymphoid tissue inducer (LTi) cells program the development of mammalian lymph nodes. In this study, we show that although primary CD4 T cell expansion is normal in RORγ-deficient mice, the persistence of memory CD4 T cells is RORγ-dependent. Furthermore, using bone marrow chimeric mice we demonstrate that LTi cells are the key RORγ-expressing cell type sufficient for memory CD4 T cell survival in the absence of persistent Ag. This effect was specific for CD4 T cells, as memory CD8 T cells survived equally well in the presence or absence of LTi cells. These data demonstrate a novel role for LTi cells, archetypal members of the innate lymphoid cell family, in supporting memory CD4 T cell survival in vivo.

The vitamin A metabolite and transcriptional modulator retinoic acid (RA) is recognized as an important regulator of epithelial cell homeostasis in several tissues. Despite the known importance of the epithelial compartment of the thymus in T cell development and selection, the potential role of RA in the regulation of thymic cortical and medullary epithelial cell homeostasis has yet to be addressed. In this study, using fetal thymus organ cultures, we demonstrate that endogenous RA signaling promotes thymic epithelial cell (TEC) cell-cycle exit and restricts TEC cellularity preferentially in the cortical TEC compartment. Combined gene expression, biochemical, and functional analyses identified mesenchymal cells as the major source of RA in the embryonic thymus. In reaggregate culture experiments, thymic mesenchyme was required for RA-dependent regulation of TEC expansion, highlighting the importance of mesenchyme-derived RA in modulating TEC turnover. The RA-generating potential of mesenchymal cells was selectively maintained within a discrete Ly51intgp38+ subset of Ly51+ mesenchyme in the adult thymus, suggesting a continual role for mesenchymal cell-derived RA in postnatal TEC homeostasis. These findings identify RA signaling as a novel mechanism by which thymic mesenchyme influences TEC development.

In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior.

FoxP3-deficient mice are rescued from tissue and organ destruction and subsequent lethal autoimmune disease by the combined absence of OX40 and CD30 signals., Our previous studies have implicated signaling through the tumor necrosis family receptors OX40 and CD30 as critical for maintaining CD4 memory responses. We show that signals through both molecules are also required for CD4 effector-mediated autoimmune tissue damage. Under normal circumstances, male mice deficient in the forkhead transcription factor FoxP3, which lack regulatory CD4 T cells, develop lethal autoimmune disease in the first few weeks of life. However, in the combined absence of OX40 and CD30, FoxP3-deficient mice develop normally and breed successfully. The extensive tissue infiltration and organ destruction characteristic of FoxP3 disease does not appear in these mice, and their mortality is not associated with autoimmunity. Although the absence of OX40 plays the dominant role, FoxP3-deficient mice sufficient in CD30 but deficient in OX40 signals still eventually develop lethal disease. This result was supported by the observation that blocking antibodies to OX40 and CD30 ligands also abrogated disease mediated by FoxP3-deficient T cells. These observations identify OX40 and CD30 signals as essential for the development of clinically relevant CD4-dependent autoimmunity and suggest that combination therapies that abrogate these signals might be used to treat established human autoimmune diseases.

Thymic epithelium provides an essential cellular substrate for T cell development and selection. Gradual age-associated thymic atrophy leads to a reduction in functional thymic tissue and a decline in de novo T cell generation. Development of strategies tailored toward regeneration of thymic tissue provides an important possibility to improve immune function in elderly individuals and increase the capacity for immune recovery in patients having undergone bone marrow transfer following immunoablative therapies. In this study we show that restriction of the size of the functional thymic epithelial progenitor pool affects the number of mature thymic epithelial cells. Using an embryo fusion chimera-based approach, we demonstrate a reduction in the total number of both embryonic and adult thymic epithelium, which relates to the initial size of the progenitor cell pool. The inability of thymic epithelial progenitor cells to undergo sufficient compensatory proliferation to rescue the deficit in progenitor numbers suggests that in addition to extrinsic regulation of thymus growth by provision of growth factors, intrinsic factors such as a proliferative restriction of thymic epithelial progenitors and availability of progenitor cell niches may limit thymic epithelial recovery. Collectively, our data demonstrate an important level of regulation of thymic growth and recovery at the thymic epithelial progenitor level, providing an important consideration for developing methods targeted toward inducing thymic regeneration.

usc Intrathymic deletion of immature thymocytes that express self-reactive TCR specificities is essential in the generation of self tolerance. Medullary thymic epithelial cells (mTEC) expressing the transcriptional regulator Aire play a key role in this process by regulating expression of tissue-restricted antigens to ensure tolerance to peripheral tissues. Here, we have analysed the cellular and molecular requirements for the initial appearance of Aire+ mTEC in the embryonic thymus, in addition to their persistence in the adult thymus. Analysis of thymic ontogeny shows that the emergence of embryonic Aire+ mTEC occurs prior to the appearance of mature thymocytes, and depends upon lymphoid tissue inducer cells expressing retinoic acid receptor-related orphan receptor γ. In the adult thymus, we show that Aire+ mTEC develop in the absence of thymocyte positive and negative selection and CD40 signalling, but are present at reduced frequency. Collectively these data support a model where the initial differentiation of Aire+ mTEC involves receptor activator of NF-κB (RANK)-RANKL interactions with lymphoid tissue inducer cells, with subsequent mTEC turnover and/or survival involving CD40-mediated signalling following interactions with mature CD4+ thymocytes that express CD40L. Refereed/Peer-reviewed

Lymph nodes provide specialized stromal microenvironments that support the recruitment and organization of T cells and B cells, enabling them to effectively participate in immune responses. While CD4+3− lymphoid tissue inducer cells (LTic's) are known to play a key role in influencing lymph node (LN) development, the mechanisms that regulate the development of stromal organizer cells are unclear. Here, we define an ontogenetic program of lymph node stromal cell maturation in relation to the requirement for LTic's. We also describe a lymph node reaggregation assay to study cell-cell interactions and lymphocyte recruitment to these organs that reproduces the in vivo events during lymph node development. In addition, analysis of the lymph node anlagen in normal and lymphotoxin a (LTa)–deficient embryos shows that LTa-mediated signaling is required to sustain proliferation and survival of stromal cells in vivo. Our data identify LTa-independent and LTa-dependent stages of lymph node development, and provide direct evidence for the role of LTic's during LN organogenesis.

In the thymus, medullary thymic epithelial cells (mTEC) regulate T cell tolerance via negative selection and Foxp3(+) regulatory T cell (Treg) development, and alterations in the mTEC compartment can lead to tolerance breakdown and autoimmunity. Both the receptor activator for NF-κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis and expression of the transcriptional regulator Aire are involved in the regulation of thymus medullary microenvironments. However, their impact on the mechanisms controlling mTEC homeostasis is poorly understood, as are the processes that enable the thymus medulla to support the balanced production of mTEC-dependent Foxp3(+) Treg. In this study, we have investigated the control of mTEC homeostasis and examined how this process impacts the efficacy of Foxp3(+) Treg development. Using newly generated RANK Venus reporter mice, we identify distinct RANK(+) subsets that reside within both the mTEC(hi) and mTEC(lo) compartments and that represent direct targets of OPG-mediated control. Moreover, by mapping OPG expression to a subset of Aire(+) mTEC, our data show how cis- and trans-acting mechanisms are able to control the thymus medulla by operating on multiple mTEC targets. Finally, we show that whereas the increase in mTEC availability in OPG-deficient (Tnfrsf11b(-/-)) mice impacts the intrathymic Foxp3(+) Treg pool by enhancing peripheral Treg recirculation back to the thymus, it does not alter the number of de novo Rag2pGFP(+)Foxp3(+) Treg that are generated. Collectively, our study defines patterns of RANK expression within the thymus medulla, and it shows that mTEC homeostasis is not a rate-limiting step in intrathymic Foxp3(+) Treg production.

We report here the quantitative expression of a set of immunity-related genes, including TNF family members, chemokine receptors, and transcription factors, in a CD4+CD3− accessory cell. By correlating gene expression between cell-sorted populations of defined phenotype, we show that the genetic fingerprint of these CD4+CD3− cells is distinct from dendritic cells, plasmacytoid dendritic cells, T cells, B cells, and NK cells. In contrast, it is highly similar to CD4+CD3− cells isolated from embryonic and neonatal tissues, with the exception that only adult populations express OX40L and CD30L. We have previously reported that IL-7 signals regulate CD30L expression. In the present study, we show that both neonatal and adult CD4+CD3− cells express the TNF family member, death receptor 3 (TNFRSF25), and that addition of TL1A (TNFSF15), the ligand for death receptor 3, up-regulates OX40L on neonatal CD4+CD3− cells. Finally, we demonstrate that this differentiation occurs in vivo: neonatal CD4+CD3− cells up-regulate both CD30L and OX40L after adoptive transfer into an adult recipient.

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.

In this report, we have examined the expression of the T cell survival signals, OX40 ligand (OX40L) and CD30 ligand (CD30L) on CD4+CD3−CD11c−B220−IL-7Rα+ inducer cells from birth to adulthood in mice. We found that adult but not neonatal inducer cells expressed high levels of OX40L and CD30L, whereas their expression of TNF-related activation-induced cytokine (TRANCE) and receptor activator of NF-κB (RANK) was comparable. The failure of neonatal inducer cells to express the ligands that rescue T cells helps to explain why exposure to Ag in neonatal life induces tolerance rather than immunity. The expression of OX40L and CD30L on inducer cells increased gradually in the first few weeks of life achieving essentially normal levels around the time mice were weaned. We found that IL-7 signaling through the common cytokine receptor γ-chain was critical for the optimal expression of both TNF-related activation-induced cytokine and CD30L but not OX40L. Furthermore, glucocorticoids, which potently suppress T effector function, did not influence the expression of OX40L and CD30L in the presence of IL-7.

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal αβT-cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC-II(low) CD40(-) cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin(+) thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1(-/-) mice. Moreover, CCRL1(-/-) mice have no major perturbations in T-cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.

αβT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αβT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αβT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αβT cell lineages, notably the induction of Foxp3+ regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αβT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αβT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire−/− mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3+ regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αβT cell lineages to access the thymic medulla.

The development of CD4(+) helper and CD8(+) cytotoxic T-cells expressing the αβ form of the T-cell receptor (αβTCR) takes place in the thymus, a primary lymphoid organ containing distinct cortical and medullary microenvironments. While the cortex represents a site of early T-cell precursor development, and the positive selection of CD4(+)8(+) thymocytes, the thymic medulla plays a key role in tolerance induction, ensuring that thymic emigrants are purged of autoreactive αβTCR specificities. In recent years, advances have been made in understanding the development and function of thymic medullary epithelial cells, most notably the subset defined by expression of the Autoimmune Regulator (Aire) gene. Here, we summarize current knowledge of the developmental mechanisms regulating thymus medulla development, and examine the role of the thymus medulla in recessive (negative selection) and dominant (T-regulatory cell) tolerance.

The development of CD4+ helper and CD8+ cytotoxic T-cells expressing the αβ form of the T-cell receptor (αβTCR) takes place in the thymus, a primary lymphoid organ containing distinct cortical and medullary microenvironments. While the cortex represents a site of early T-cell precursor development, and the positive selection of CD4+8+ thymocytes, the thymic medulla plays a key role in tolerance induction, ensuring that thymic emigrants are purged of autoreactive αβTCR specificities. In recent years, advances have been made in understanding the development and function of thymic medullary epithelial cells, most notably the subset defined by expression of the Autoimmune Regulator (Aire) gene. Here, we summarize current knowledge of the developmental mechanisms regulating thymus medulla development, and examine the role of the thymus medulla in recessive (negative selection) and dominant (T-regulatory cell) tolerance.

T cell tolerance in the thymus is a key step in shaping the developing T cell repertoire. Thymic medullary epithelial cells play multiple roles in this process, including negative selection of autoreactive thymocytes, influencing thymic dendritic cell positioning, and the generation of Foxp3+ regulatory T cells. Previous studies show that medullary thymic epithelial cell (mTEC) development involves hemopoietic cross-talk, and numerous TNFR superfamily members have been implicated in this process. Whereas CD40 and RANK represent key examples, interplay between these receptors, and the individual cell types providing their ligands at both fetal and adult stages of thymus development, remain unclear. In this study, by analysis of the cellular sources of receptor activator for NF-κB ligand (RANKL) and CD40L during fetal and adult cross-talk in the mouse, we show that the innate immune cell system drives initial fetal mTEC development via expression of RANKL, but not CD40L. In contrast, cross-talk involving the adaptive immune system involves both RANKL and CD40L, with analysis of distinct subsets of intrathymic CD4+ T cells revealing a differential contribution of CD40L by conventional, but not Foxp3+ regulatory, T cells. We also provide evidence for a stepwise involvement of TNFRs in mTEC development, with CD40 upregulation induced by initial RANK signaling subsequently controlling proliferation within the mTEC compartment. Collectively, our findings show how multiple hemopoietic cell types regulate mTEC development through differential provision of RANKL/CD40L during ontogeny, revealing molecular differences in fetal and adult hemopoietic cross-talk. They also suggest a stepwise process of mTEC development, in which RANK is a master player in controlling the availability of other TNFR family members.

Thymic atrophy is a frequent consequence of infection with bacteria, viruses, and parasites and is considered a common virulence trait between pathogens. Multiple reasons have been proposed to explain this atrophy, including premature egress of immature thymocytes, increased apoptosis, or thymic shutdown to prevent tolerance to the pathogen from developing. The severe loss in thymic cell number can reflect an equally dramatic reduction in thymic output, potentially reducing peripheral T cell numbers. In this study, we examine the relationship between systemic Salmonella infection and thymic function. During infection, naive T cell numbers in peripheral lymphoid organs increase. Nevertheless, this occurs despite a pronounced thymic atrophy caused by viable bacteria, with a peak 50-fold reduction in thymocyte numbers. Thymic atrophy is not dependent upon homeostatic feedback from peripheral T cells or on regulation of endogenous glucocorticoids, as demonstrated by infection of genetically altered mice. Once bacterial numbers fall, thymocyte numbers recover, and this is associated with increases in the proportion and proliferation of early thymic progenitors. During atrophy, thymic T cell maturation is maintained, and single-joint TCR rearrangement excision circle analysis reveals there is only a modest fall in recent CD4+ thymic emigrants in secondary lymphoid tissues. Thus, thymic atrophy does not necessarily result in a matching dysfunctional T cell output, and thymic homeostasis can constantly adjust to systemic infection to ensure that naive T cell output is maintained.

The role of mesenchymal stromal cells (MSCs) in regulating immune responses in the thymus is currently unclear. Here we report the existence and role of a MSC population in the thymus that expresses the pericyte and MSC marker CD248 (endosialin). We show using a CD248-deficient mouse model, that CD248 expression on these cells is required for full post-natal thymus development and regeneration post-Salmonella infection. In CD248−/− mice the thymus is hypocellular and regeneration is poorer, with significant loss of all thymocyte populations. This identifies the requirement of CD248 to maintain optimal thymic cellularity post-partum and infection., Highlights ▸ The mesenchymal stromal cell marker CD248 is expressed in the thymus on pericytes. ▸ Genetic deletion of CD248 prevents full post-natal development of the thymus. ▸ Genetic deletion of CD248 prevents infection-dependent regeneration following thymic atrophy. ▸ CD248 regulates blood endothelial vessel formation in the thymus.

The thymus imparts a developmental imprint upon T cells, screening beneficial and self-tolerant T cell receptor (TCR) specificities. Cortical thymic epithelial cells (CTEC) present self-peptide self-MHC complexes to thymocytes, positively selecting those with functional TCRs. Importantly, CTEC generate diverse self-peptides through highly specific peptide processing. The array of peptides utilized for positive selection appears to play a key role in shaping TCR repertoire and influencing T cell functionality. Whilst self-peptide diversity influences T cell development, the precise source of proteins generating such self-peptide arrays remains unknown, the abundance of apoptotic thymocytes failing thymic selection may provide such a pool of self-proteins. In relation to this notion, whilst it has been previously demonstrated that CTEC expression of the endocytic receptor CD205 facilitates binding and uptake of apoptotic thymocytes, the possible role of CD205 during intrathymic T cell development has not been studied. Here, we directly address the role of CD205 in normal thymocyte development and selection. Through analysis of both polyclonal and monoclonal transgenic TCR T-cell development in the context of CD205 deficiency, we demonstrate that CD205 does not play an overt role in T cell development or selection.

SummaryLymph 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.