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By using high-throughput sequencing of T-cell receptors, this study shows that thymus-derived regulatory T (T reg ) cells constitute most T reg cells in all lymphoid and intestinal organs, including the colon, suggesting that thymic T reg cells and not induced T reg cells dominantly control tolerance to the gut’s antigens such as commensal microbiota. T cells and commensal microbe tolerance The identity of the intestinal regulatory T (T reg ) cells that control autoimmune diseases such as colitis and immune responses to commensal microbes remains ill defined. In this study Anna Cebula et al . use high-throughput sequencing of T-cell receptors to show that the predominant T reg cells in all lymphoid and intestinal organs, including the colon, are derived from the thymus. This challenges the view that induced rather than thymic T reg cells are mainly responsible for the control of intestinal inflammation, although it does not exclude the possibility that induced T reg cells contribute to intestinal homeostasis. Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4 + Foxp3 + regulatory T (T reg ) cells 1 , 2 generated in the thymus or extrathymically by induction of naive CD4 + Foxp3 − T cells. Previous studies suggested that the T-cell receptor repertoires of thymic T reg cells and induced T reg cells are biased towards self and non-self antigens, respectively 3 , 4 , 5 , 6 , but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved 7 . The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which T reg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage 8 , 9 and favour tolerogenic presentation of antigens to naive CD4 + T cells 10 , 11 , suggesting that intestinal homeostasis depends on microbiota-specific induced T reg cells 12 , 13 , 14 , 15 . Here, to identify the origin and antigen-specificity of intestinal T reg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4 + Foxp3 + and CD4 + Foxp3 − T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived T reg cells constitute most T reg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic T reg cells, and not induced T reg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.

The thymus is essential for establishing adaptive immunity yet undergoes age-related involution that leads to compromised immune responsiveness. The thymus is also extremely sensitive to acute insult and although capable of regeneration, this capacity declines with age for unknown reasons. We applied single-cell and spatial transcriptomics, lineage-tracing and advanced imaging to define age-related changes in nonhematopoietic stromal cells and discovered the emergence of two atypical thymic epithelial cell (TEC) states. These age-associated TECs (aaTECs) formed high-density peri-medullary epithelial clusters that were devoid of thymocytes; an accretion of nonproductive thymic tissue that worsened with age, exhibited features of epithelial-to-mesenchymal transition and was associated with downregulation of FOXN1. Interaction analysis revealed that the emergence of aaTECs drew tonic signals from other functional TEC populations at baseline acting as a sink for TEC growth factors. Following acute injury, aaTECs expanded substantially, further perturbing trophic regeneration pathways and correlating with defective repair of the involuted thymus. These findings therefore define a unique feature of thymic involution linked to immune aging and could have implications for developing immune-boosting therapies in older individuals. Here the authors identify age-associated changes in the epithelial cell compartment of the thymus that form high-density nonproductive microenvironmental niches that contribute toward thymic involution and inhibit its repair following injury.

The differentiation of αβ T cells is a complex process. Using data sets from the Immunological Genome Project, Benoist and colleagues identify candidate mediators of key transitions during thymocyte selection and maturation. The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4 + CD8 + stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4 + or CD8 + lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

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 mTEC low 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 + mTEC low 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.

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR–pMHC–CD8 trimolecular ‘catch bonds’, whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR–pMHC and pMHC–CD8 bimolecular ‘slip bonds’. Catch bonds were not intrinsic to either the TCR–pMHC or the pMHC–CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC–CD8 interaction to the intracellular interaction of CD8 with TCR–CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans–cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus. Zhu and colleagues show that negative-selection ligands induce cooperative TCR–pMHC–CD8 trimolecular ‘catch bonds’, whereas positive-selection ligands induce TCR–pMHC and pMHC–CD8 bimolecular ‘slip bonds’.

Even though the thymus is exquisitely sensitive to acute insults like infection, shock, or common cancer therapies such as cytoreductive chemo- or radiation-therapy, it also has a remarkable capacity for repair. This phenomenon of endogenous thymic regeneration has been known for longer even than its primary function to generate T cells, however, the underlying mechanisms controlling the process have been largely unstudied. Although there is likely continual thymic involution and regeneration in response to stress and infection in otherwise healthy people, acute and profound thymic damage such as that caused by common cancer cytoreductive therapies or the conditioning regimes as part of hematopoietic cell transplantation (HCT), leads to prolonged T cell deficiency; precipitating high morbidity and mortality from opportunistic infections and may even facilitate cancer relapse. Furthermore, this capacity for regeneration declines with age as a function of thymic involution; which even at steady state leads to reduced capacity to respond to new pathogens, vaccines, and immunotherapy. Consequently, there is a real clinical need for strategies that can boost thymic function and enhance T cell immunity. One approach to the development of such therapies is to exploit the processes of endogenous thymic regeneration into novel pharmacologic strategies to boost T cell reconstitution in clinical settings of immune depletion such as HCT. In this review, we will highlight recent work that has revealed the mechanisms by which the thymus is capable of repairing itself and how this knowledge is being used to develop novel therapies to boost immune function.

Clonal deletion through negative selection is critical to eliminate autoreactive T cells in the thymus. Negative selection, however, is imperfect such that some autoreactive thymocytes can escape thymic deletion and successfully populate peripheral tissues. This is also the case for autoreactive 2D2 TCR transgenic T cells, a widely employed mouse model in studying the pathogenesis of CD4 T cell-mediated experimental autoimmune encephalomyelitis. How autoreactive 2D2 thymocytes evade negative selection, however, remains incompletely understood. Here we show that negative selection of MHC-II-restricted thymocytes, specifically 2D2 TCR transgenic T cells, is associated with the induction of integrin CD103, and that forced expression of CD103 downregulates CXCR4 expression, alters intra-thymic trafficking, and reinforces clonal deletion of immature thymocytes. Stratification of positively versus negatively selected 2D2 T cells based on their distinct coreceptor expression further shows that CD103 does not affect the generation of conventional CD4 T cells but is deleterious for autoreactive CD4, CD8 double-negative 2D2 T cells that correspond to CD69-negative CCR7-intermediate thymocytes, displaying markers of agonistic TCR signalling. Collectively, these results propose CD103 expression as an indicator and contributor of negative selection for MHC-II-restricted T cells, providing further mechanistic insights into the process of T cell selection in the thymus. Clonal deletion is an important mechanism for the elimination of autoreactive T cells, however, negative selection of thymocytes is imperfect. Here authors show that MHC-II-restricted thymocytes avoid negative selection via downregulation of the integrin CD103, altering intra-thymic trafficking and distribution in a way that favours their survival.

Females exhibit a more robust immune response to both self-antigens and non-self-antigens than males, resulting in a higher prevalence of autoimmune diseases but more effective responses against infection. Increased expression of X-linked immune genes in female T cells is thought to underlie this enhanced response. Here we isolate thymocytes from pediatric thymi of healthy males (46, XY), females (46, XX), a female with completely skewed X-chromosome inactivation (46, XX, cXCI) and a female with Turner syndrome (45, X0). Using whole exome sequencing, RNA sequencing and DNA methylation data, we present a sex-aware expression profile of T cell development and generate a high-resolution map of escape from X-chromosome inactivation (XCI). Unexpectedly, XCI is transcriptionally and epigenetically stable throughout T cell development, and is independent of expression of XIST , the lncRNA responsible for XCI initiation during early embryonic development. In thymocytes, several genes known to escape XCI are expressed from only one X-chromosome. Additionally, we further reveal that a second X-chromosome is dispensable for T cell development. Our study thus provides a high-resolution map of XCI during human development and suggests a re-evaluation of XCI in sex differences in T cell function. X-chromosome inactivation (XCI) contributes to sex bias in T cell immunity, but data on profiling XCI during human T cell development is still lacking. Here, the authors leverage allele-specific expression, sex-biased gene expression and DNA methylation data on human pediatric thymocytes to find surprisingly stable XCI during thymocyte differentiation.

Mucosal-associated invariant T cells (MAIT cells) recognize the microbial metabolite 5-(2-oxopropylideneamino)-6- d -ribitylaminouracil (5-OP-RU) presented by the MHC class Ib molecule, MR1. MAIT cells acquire effector functions during thymic development, but the mechanisms involved are unclear. Here we used single-cell RNA-sequencing to characterize the developmental path of 5-OP-RU-specific thymocytes. In addition to the known MAIT1 and MAIT17 effector subsets selected on bone-marrow-derived hematopoietic cells, we identified 5-OP-RU-specific thymocytes that were selected on thymic epithelial cells and differentiated into CD44 − naive T cells. MAIT cell positive selection required signaling through the adapter, SAP, that controlled the expression of the transcription factor, ZBTB16. Pseudotemporal ordering of single cells revealed transcriptional trajectories of 5-OP-RU-specific thymocytes selected on either thymic epithelial cells or hematopoietic cells. The resulting model illustrates T cell lineage decisions. MAIT cells recognize the microbial metabolite 5-OP-RU and are selected on DP thymocytes expressing the MHC class Ib molecule MR1. Lantz and colleagues identify 5-OP-RU-specific thymocytes that are selected on thymic epithelial cells and differentiate into naive T cells.

The transcription factor NF-κB is required for T cell effector function. Gerondakis and colleagues discuss the role of NF-κB in T cell development. The NF-κB signal transduction pathway is best known as a major regulator of innate and adaptive immune responses, yet there is a growing appreciation of its importance in immune cell development, particularly of T lineage cells. In this Review, we discuss how the temporal regulation of NF-κB controls the stepwise differentiation and antigen-dependent selection of conventional and specialized subsets of T cells in response to T cell receptor and costimulatory, cytokine and growth factor signals.