Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

Progress on thymic function from Maui.

Maui, Hawaii, was the home for the latest Global Thymus Network meeting 5-9 June 2016. Participants gathered together to discuss how one little organ can have such huge effects.

Trust your gut: an early life lesson for T cells.

Zegarra-Ruiz et al. demonstrate that gut microbe-specific T cells are selected within the thymus exclusively during early life in mice. This selection is dependent on CX3CR1+ dendritic cells migrating from the gut carrying bacterially derived antigens into the thymus. This offers new insight into how gut microbiota influence T cell development.

Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk.

In the thymus, cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells support αßT cell development from lymphoid progenitors. For cTECs, expression of a specialized gene signature that includes Cxcl12, Dll4, and Psmb11 enables the cortex to support T lineage commitment and the generation and selection of CD4+CD8+ thymocytes. Although the importance of cTECs in T cell development is well defined, mechanisms that shape the cTEC compartment and regulate its functional specialization are unclear. Using a Cxcl12 DsRed reporter mouse model, we show that changes in Cxcl12 expression reveal a developmentally regulated program of cTEC heterogeneity. Although cTECs are uniformly Cxcl12 DsRed+ during neonatal stages, progression through postnatal life triggers the appearance of Cxcl12 DsRed- cTECs that continue to reside in the cortex alongside their Cxcl12 DsRed+ counterparts. This appearance of Cxcl12 DsRed- cTECs is controlled by maturation of CD4-CD8-, but not CD4+CD8+, thymocytes, demonstrating that stage-specific thymocyte cross-talk controls cTEC heterogeneity. Importantly, although fate-mapping experiments show both Cxcl12 DsRed+ and Cxcl12 DsRed- cTECs share a common Foxn1 + cell origin, RNA sequencing analysis shows Cxcl12 DsRed- cTECs no longer express Foxn1, which results in loss of the FOXN1-dependent cTEC gene signature and may explain the reduced capacity of Cxcl12 DsRed- cTECs for thymocyte interactions. In summary, our study shows that shaping of the cTEC compartment during the life course occurs via stage-specific thymocyte cross-talk, which drives loss of Foxn1 expression and its key target genes, which may then determine the functional competence of the thymic cortex.

Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk.

In the thymus, cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells support αßT cell development from lymphoid progenitors. For cTECs, expression of a specialized gene signature that includes Cxcl12, Dll4, and Psmb11 enables the cortex to support T lineage commitment and the generation and selection of CD4+CD8+ thymocytes. Although the importance of cTECs in T cell development is well defined, mechanisms that shape the cTEC compartment and regulate its functional specialization are unclear. Using a Cxcl12DsRed reporter mouse model, we show that changes in Cxcl12 expression reveal a developmentally regulated program of cTEC heterogeneity. Although cTECs are uniformly Cxcl12DsRed+ during neonatal stages, progression through postnatal life triggers the appearance of Cxcl12DsRed- cTECs that continue to reside in the cortex alongside their Cxcl12DsRed+ counterparts. This appearance of Cxcl12DsRed- cTECs is controlled by maturation of CD4-CD8-, but not CD4+CD8+, thymocytes, demonstrating that stage-specific thymocyte cross-talk controls cTEC heterogeneity. Importantly, although fate-mapping experiments show both Cxcl12DsRed+ and Cxcl12DsRed- cTECs share a common Foxn1+ cell origin, RNA sequencing analysis shows Cxcl12DsRed- cTECs no longer express Foxn1, which results in loss of the FOXN1-dependent cTEC gene signature and may explain the reduced capacity of Cxcl12DsRed- cTECs for thymocyte interactions. In summary, our study shows that shaping of the cTEC compartment during the life course occurs via stage-specific thymocyte cross-talk, which drives loss of Foxn1 expression and its key target genes, which may then determine the functional competence of the thymic cortex.

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis.

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth.

Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells.

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.

Aire controls the recirculation of murine Foxp3+ regulatory T-cells back to the thymus.

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4+ and CD8+ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self-antigen availability for negative selection. Interestingly, alterations in both Foxp3+ Regulatory T-cells (T-Reg) and conventional SP thymocytes in Aire-/- mice suggest additional, yet poorly understood, roles for Aire during intrathymic T-cell development. To examine this, we analysed thymocytes from Aire-/- mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post-selection CD4+ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αßT-cells, it plays a key role in controlling the intrathymic T-Reg pool. Surprisingly, a decline in intrathymic T-Reg in Aire-/- mice maps to a reduction in mature recirculating Rag2GFP- T-Reg that express CCR6 and re-enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire-/- mice, and that CCR6 is required for T-Reg recirculation back to the thymus. Collectively, our study re-defines requirements for late stage intrathymic αßT-cell development, and demonstrates that Aire controls a CCR6-CCL20 axis that determines the developmental makeup of the intrathymic T-Reg pool.

Osteoprotegerin-Mediated Homeostasis of Rank+ Thymic Epithelial Cells Does Not Limit Foxp3+ Regulatory T Cell Development.

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.

Thymus medulla fosters generation of natural Treg cells, invariant γδ T cells, and invariant NKT cells: what we learn from intrathymic migration.

The organization of the thymus into distinct cortical and medullary regions enables it to control the step-wise migration and development of immature T-cell precursors. Such a process provides access to specialized cortical and medullary thymic epithelial cells at defined stages of maturation, ensuring the generation of self-tolerant and MHC-restricted conventional CD4(+) and CD8(+) αß T cells. The migratory cues and stromal cell requirements that regulate the development of conventional αß T cells have been well studied. However, the thymus also fosters the generation of several immunoregulatory T-cell populations that form key components of both innate and adaptive immune responses. These include Foxp3(+) natural regulatory T cells, invariant γδ T cells, and CD1d-restricted invariant natural killer T cells (iNKT cells). While less is known about the intrathymic requirements of these nonconventional T cells, recent studies have highlighted the importance of the thymus medulla in their development. Here, we review recent findings on the mechanisms controlling the intrathymic migration of distinct T-cell subsets, and relate this to knowledge of the microenvironmental requirements of these cells.

An essential role for medullary thymic epithelial cells during the intrathymic development of invariant NKT cells.

In the thymus, interactions with both cortical and medullary microenvironments regulate the development of self-tolerant conventional CD4(+) and CD8(+) αßT cells expressing a wide range of αßTCR 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 αßTCR 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 αßT 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.

Differential requirement for CCR4 and CCR7 during the development of innate and adaptive αßT cells in the adult thymus.

αßT 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 αßT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αßT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αßT cell lineages, notably the induction of Foxp3(+) regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αßT 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 αßT 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 αßT cell lineages to access the thymic medulla.

Natural Th17 cells are critically regulated by functional medullary thymic microenvironments.

The thymic medulla is critical for the enforcement of central tolerance. In addition to deletion of auto-reactive T-cells, the thymic medulla supports the maturation of heterogeneous natural αßT-cells linked to tolerance mechanisms. Natural IL-17-secreting CD4(+)αßT-cells (nTh17) represent recently described natural αßT-cells that mature and undergo functional priming intrathymically. Despite a proposed potential to impact upon either protective or pathological inflammatory responses, the intrathymic mechanisms regulating the balance of nTh17 development are unclear. Here we compare the development of distinct natural αßT-cells in the thymus. We reveal that thymic stromal MHC class II expression and RelB-dependent medullary thymic epithelial cells (mTEC), including Aire(+) mTEC, are an essential requirement for nTh17 development. nTh17 demonstrate a partial, non-redundant requirement for both ICOS-ligand and CD80/86 costimulation, with a dispensable role for CD80/86 expression by thymic epithelial cells. Although mTEC constitutively expressed inducible nitric oxide synthase (iNOS), a critical negative regulator of conventional Th17 differentiation, iNOS was not essential to constrain thymic nTh17. These findings highlight the critical role of the thymic medulla in the differential regulation of novel natural αßT-cell subsets, and reveal additional layers of thymic medullary regulation of T-cell driven autoimmunity and inflammation.

Mechanisms of thymus medulla development and function.

The development of CD4(+) helper and CD8(+) cytotoxic T-cells expressing the αß form of the T-cell receptor (αßTCR) 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 αßTCR 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.

Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla.

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.

Revelations in Thymic Epithelial Cell Biology and Heterogeneity from Single-Cell RNA Sequencing and Lineage Tracing Methodologies.

Thymic epithelial cells (TECs) make up the thymic microenvironments that support the generation of a functionally competent and self-tolerant T-cell repertoire. Cortical (c)TECs, present in the cortex, are essential for early thymocyte development including selection of thymocytes expressing functional TCRs (positive selection). Medullary (m)TECs, located in the medulla, play a key role in late thymocyte development, including depletion of self-reactive T cells (negative selection) and selection of regulatory T cells. In recent years, transcriptomic analysis by single-cell (sc)RNA sequencing (Seq) has revealed TEC heterogeneity previously masked by population-level RNA-Seq or phenotypic studies. We summarize the discoveries made possible by scRNA-Seq, including the identification of novel mTEC subsets, advances in understanding mTEC promiscuous gene expression, and TEC alterations from embryonic to adult stages. Whereas pseudotime analyses of scRNA-Seq data can suggest relationships between TEC subsets, experimental methods such as lineage tracing and reaggregate thymic organ culture (RTOC) are required to test these hypotheses. Lineage tracing - namely, of ß5t or Aire expressing cells - has exposed progenitor and parent-daughter cellular relationships within TEC.

Expression of the transcription factor Klf6 by thymic epithelial cells is required for thymus development.

Thymic epithelial cells (TEC) control T cell development and play essential roles in establishing self-tolerance. By using Foxn1-Cre-driven ablation of Klf6 gene in TEC, we identified Klf6 as a critical factor in TEC development. Klf6 deficiency resulted in a hypoplastic thymus-evident from fetal stages into adulthood-in which a dramatic increase in the frequency of apoptotic TEC was observed. Among cortical TEC (cTEC), a previously unreported cTEC population expressing the transcription factor Sox10 was relatively expanded. Within medullary TEC (mTEC), mTEC I and Tuft-like mTEC IV were disproportionately decreased. Klf6 deficiency altered chromatin accessibility and affected TEC chromatin configuration. Consistent with these defects, naïve conventional T cells and invariant natural killer T cells were reduced in the spleen. Late stages of T cell receptor-dependent selection of thymocytes were affected, and mice exhibited autoimmunity. Thus, Klf6 has a prosurvival role and affects the development of specific TEC subsets contributing to thymic function.

Process evaluation of an environmental and educational nutrition intervention in residential drug-treatment facilities.

OBJECTIVE: To evaluate the implementation of a controlled, 6-week, environmental and educational intervention to improve dietary intake and body composition, and to study the association of implementation fidelity with diet and body composition outcomes. DESIGN: A process evaluation documented participation, dose of nutrition education delivered, participant satisfaction, fidelity and completeness of the food environment intervention implementation, and context through observations and interviews with staff and residents. Intervention sites were scored and categorized as high or low participation and implementation and compared on essential elements of the food environment and on diet and body composition outcomes. SETTING: Six urban residential drug-treatment facilities in Upstate New York. SUBJECTS: Fifty-five primarily black and white men in residential drug-treatment programmes. RESULTS: Participants were exposed to 94 % and 69 % of the educational and environmental elements, respectively. High implementation sites were significantly more likely to provide water and 100 % juice, offer fruit or vegetable salad, offer choices of fruits and vegetables, and limit fried foods. Mixed-model analysis of covariance revealed that participants in the high participation and implementation sites reported greater reductions in total energy, percentage of energy from sweets, daily servings of fats, oils and sweets, and BMI over the intervention period. Participants in low participation and implementation sites reported greater reductions in percentage of energy from fat. Differential implementation of environmental elements limited the intervention impact. CONCLUSIONS: These findings document the contribution of changes in eating environments to facilitate dietary behaviour change in community residential substance-abuse settings.

Postnatal Involution and Counter-Involution of the Thymus.

Thymus involution occurs in all vertebrates. It is thought to impact on immune responses in the aged, and in other clinical circumstances such as bone marrow transplantation. Determinants of thymus growth and size are beginning to be identified. Ectopic expression of factors like cyclin D1 and Myc in thymic epithelial cells (TEC)s results in considerable increase in thymus size. These models provide useful experimental tools that allow thymus function to be understood. In future, understanding TEC-specific controllers of growth will provide new approaches to thymus regeneration.