Let-7 enhances murine anti-tumor CD8 T cell responses by promoting memory and antagonizing terminal differentiation.

The success of the CD8 T cell-mediated immune response against infections and tumors depends on the formation of a long-lived memory pool, and the protection of effector cells from exhaustion. The advent of checkpoint blockade therapy has significantly improved anti-tumor therapeutic outcomes by reversing CD8 T cell exhaustion, but fails to generate effector cells with memory potential. Here, using in vivo mouse models, we show that let-7 miRNAs determine CD8 T cell fate, where maintenance of let-7 expression during early cell activation results in memory CD8 T cell formation and tumor clearance. Conversely, let-7-deficiency promotes the generation of a terminal effector population that becomes vulnerable to exhaustion and cell death in immunosuppressive environments and fails to reject tumors. Mechanistically, let-7 restrains metabolic changes that occur during T cell activation through the inhibition of the PI3K/AKT/mTOR signaling pathway and production of reactive oxygen species, potent drivers of terminal differentiation and exhaustion. Thus, our results reveal a role for let-7 in the time-sensitive support of memory formation and the protection of effector cells from exhaustion. Overall, our data suggest a strategy in developing next-generation immunotherapies by preserving the multipotency of effector cells rather than enhancing the efficacy of differentiation.

Combined multidimensional single-cell protein and RNA profiling dissects the cellular and functional heterogeneity of thymic epithelial cells.

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T cell development and selection. Recent single-cell RNA sequencing studies have uncovered previously unappreciated transcriptional heterogeneity among thymic epithelial cells (TEC). However, there are only very few cell markers that allow a comparable phenotypic identification of TEC. Here, using massively parallel flow cytometry and machine learning, we deconvoluted known TEC phenotypes into novel subpopulations. Using CITEseq, these phenotypes were related to corresponding TEC subtypes defined by the cells' RNA profiles. This approach allowed the phenotypic identification of perinatal cTEC and their physical localisation within the cortical stromal scaffold. In addition, we demonstrate the dynamic change in the frequency of perinatal cTEC in response to developing thymocytes and reveal their exceptional efficiency in positive selection. Collectively, our study identifies markers that allow for an unprecedented dissection of the thymus stromal complexity, as well as physical isolation of TEC populations and assignment of specific functions to individual TEC subtypes.

IL-2 and IL-15 drive intrathymic development of distinct periphery-seeding CD4+Foxp3+ regulatory T lymphocytes.

Development of Foxp3-expressing regulatory T-lymphocytes (Treg) in the thymus is controlled by signals delivered in T-cell precursors via the TCR, co-stimulatory receptors, and cytokine receptors. In absence of IL-2, IL-15 or their receptors, fewer Treg apparently develop in the thymus. However, it was recently shown that a substantial part of thymic Treg are cells that had recirculated from the periphery back to the thymus, troubling interpretation of these results. We therefore reassessed the involvement of IL-2 and IL-15 in the development of Treg, taking into account Treg-recirculation. At the age of three weeks, when in wt and IL-15-deficient (but not in IL-2-deficient) mice substantial amounts of recirculating Treg are present in the thymus, we found similarly reduced proportions of newly developed Treg in absence of IL-2 or IL-15, and in absence of both cytokines even less Treg developed. In neonates, when practically no recirculating Treg were found in the thymus, the absence of IL-2 led to substantially more reduced Treg-development than deficiency in IL-15. IL-2 but not IL-15 modulated the CD25, GITR, OX40, and CD73-phenotypes of the thymus-egress-competent and periphery-seeding Treg-population. Interestingly, IL-2 and IL-15 also modulated the TCR-repertoire expressed by developing Treg. Upon transfer into Treg-less Foxp3sf mice, newly developed Treg from IL-2- (and to a much lesser extent IL-15-) deficient mice suppressed immunopathology less efficiently than wt Treg. Taken together, our results firmly establish important non-redundant quantitative and qualitative roles for IL-2 and, to a lesser extent, IL-15 in intrathymic Treg-development.

Abrogation of Notch Signaling in Embryonic TECs Impacts Postnatal mTEC Homeostasis and Thymic Involution.

Notch signaling is crucial for fate specification and maturation of thymus-seeding progenitors along the T-cell lineage. Recent studies have extended the role of Notch signaling to thymic epithelial cells (TECs), showing that Notch regulates TEC progenitor maintenance and emergence of medullary TECs (mTECs) in fetal thymopoiesis. Based on immunohistochemistry studies of spatiotemporal regulation of Notch activation in the postnatal thymus, we show that in vivo Notch activation is not confined to fetal TECs. Rather, Notch signaling, likely mediated through the Notch1 receptor, is induced in postnatal cortical and medullary TECs, and increases significantly with age in the latter, in both humans and mice, suggesting a conserved role for Notch signaling in TEC homeostasis during thymus aging. To investigate the functional impact of Notch activation in postnatal TEC biology, we used a mouse model in which RPBJκ, the transcriptional effector of canonical Notch signaling, is deleted in epithelial cells, including TECs, under the control of the transcription factor Foxn1. Immunohistochemistry and flow cytometry analyses revealed no significant differences in TEC composition in mutant (RPBJκ-KOTEC) and wild-type (WT) littermate mice at early postnatal ages. However, a significant reduction of the medullary region was observed in mutant compared to WT older thymi, which was accompanied by an accelerated decrease of postnatal mTEC numbers. Also, we found that organization and integrity of the postnatal thymic medulla critically depends on activation of the canonical Notch signaling pathway, as abrogation of Notch signaling in TECs led to the disruption of the medullary thymic microenvironment and to an accelerated thymus atrophy. These features paralleled a significant increase in the proportion of intrathymic non-T lineage cells, mostly B cells, and a slight decrease of DP thymocyte numbers compatible with a compromised thymic function in mutant mice. Therefore, impaired Notch signaling induced in embryonic development impacts postnatal TECs and leads to an accelerated mTEC degeneration and a premature thymus involution. Collectively, our data have uncovered a new role for Notch1 signaling in the control of adult mTEC homeostasis, and point toward Notch signaling manipulation as a novel strategy for thymus regeneration and functional recovery from immunosenescence.

FOXN1 forms higher-order nuclear condensates displaced by mutations causing immunodeficiency.

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.

Indispensable epigenetic control of thymic epithelial cell development and function by polycomb repressive complex 2.

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.

Comprehensively Profiling the Chromatin Architecture of Tissue Restricted Antigen Expression in Thymic Epithelial Cells Over Development.

Thymic epithelial cells (TEC) effect crucial roles in thymopoiesis including the control of negative thymocyte selection. This process depends on their capacity to express promiscuously genes encoding tissue-restricted antigens. This competence is accomplished in medullary TEC (mTEC) in part by the presence of the transcriptional facilitator AutoImmune REgulator, AIRE. AIRE-regulated gene transcription is marked by repressive chromatin modifications, including H3K27me3. When during TEC development these chromatin marks are established, however, remains unclear. Here we use a comprehensive ChIP-seq dataset of multiple chromatin modifications in different TEC subtypes to demonstrate that the chromatin landscape is established early in TEC differentiation. Much of the chromatin architecture found in mature mTEC was found to be present already over earlier stages of mTEC lineage differentiation as well as in non-TEC tissues. This was reflected by the fact that a machine learning approach accurately classified genes as AIRE-induced or AIRE-independent both in immature and mature mTEC. Moreover, analysis of TEC specific enhancer elements identified candidate transcription factors likely to be important in mTEC development and function. Our findings indicate that the mature mTEC chromatin landscape is laid down early in mTEC differentiation, and that AIRE is not required for large-scale re-patterning of chromatin in mTEC.

Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells.

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

Dynamic spatio-temporal contribution of single ß5t+ cortical epithelial precursors to the thymus medulla.

Intrathymic T-cell development is critically dependent on cortical and medullary thymic epithelial cells (TECs). Both epithelial subsets originate during early thymus organogenesis from progenitor cells that express the thymoproteasome subunit ß5t, a typical feature of cortical TECs. Using in vivo lineage fate mapping, we demonstrate in mice that ß5t(+) TEC progenitors give rise to the medullary TEC compartment early in life but significantly limit their contribution once the medulla has completely formed. Lineage-tracing studies at single cell resolution demonstrate for young mice that the postnatal medulla is expanded from individual ß5t(+) cortical progenitors located at the cortico-medullary junction. These results therefore not only define a developmental window during which the expansion of medulla is efficiently enabled by progenitors resident in the thymic cortex, but also reveal the spatio-temporal dynamics that control the growth of the thymic medulla.

Adult Thymic Medullary Epithelium Is Maintained and Regenerated by Lineage-Restricted Cells Rather Than Bipotent Progenitors.

Medullary thymic epithelial cells (mTECs) play an essential role in establishing self-tolerance in T cells. mTECs originate from bipotent TEC progenitors that generate both mTECs and cortical TECs (cTECs), although mTEC-restricted progenitors also have been reported. Here, we report in vivo fate-mapping analysis of cells that transcribe ß5t, a cTEC trait expressed in bipotent progenitors, during a given period in mice. We show that, in adult mice, most mTECs are derived from progenitors that transcribe ß5t during embryogenesis and the neonatal period up to 1 week of age. The contribution of adult ß5t(+) progenitors was minor even during injury-triggered regeneration. Our results further demonstrate that adult mTEC-restricted progenitors are derived from perinatal ß5t(+) progenitors. These results indicate that the adult thymic medullary epithelium is maintained and regenerated by mTEC-lineage cells that pass beyond the bipotent stage during early ontogeny.

A regulatory role for TGF-ß signaling in the establishment and function of the thymic medulla.

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-ß in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-ß signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.

TRAF3 enforces the requirement for T cell cross-talk in thymic medullary epithelial development.

Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra(-/-) mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTßR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.

Aire-expressing thymic medullary epithelial cells originate from ß5t-expressing progenitor cells.

The thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. The thymic cortex induces the generation and positive selection of T lymphocytes, whereas the thymic medulla establishes self-tolerance among the positively selected T lymphocytes. Cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively. cTECs and mTECs are both derived from the endodermal epithelium of the third pharyngeal pouch. However, the molecular and cellular characteristics of the progenitor cells for the distinct TEC lineages are unclear. Here we report the preparation and characterization of mice that express the recombinase Cre instead of ß5t, a proteasome subunit that is abundant in cTECs and not detected in other cell types, including mTECs. By crossing ß5t-Cre knock-in mice with loxP-dependent GFP reporter mice, we found that ß5t-Cre-mediated recombination occurs specifically in TECs but not in any other cell types in the mouse. Surprisingly, in addition to cTECs, ß5t-Cre-loxP-mediated GFP expression was detected in almost all mTECs. These results indicate that the majority of mTECs, including autoimmune regulator-expressing mTECs, are derived from ß5t-expressing progenitor cells.

IL-7 produced by thymic epithelial cells plays a major role in the development of thymocytes and TCRγδ+ intraepithelial lymphocytes.

IL-7 is a cytokine essential for T cell development and survival. However, the local function of IL-7 produced by thymic epithelial cells (TECs) is poorly understood. To address this question, we generated IL-7-floxed mice and crossed them with FoxN1 promoter-driven Cre (FoxN1-Cre) mice to establish knockout mice conditionally deficient for the expression of IL-7 by TECs. We found that αß and γδ T cells were significantly reduced in the thymus of IL-7(f/f) FoxN1-Cre mice. Proportion of mature single-positive thymocytes was increased. In lymph nodes and the spleen, the numbers of T cells were partially restored in IL-7(f/f) FoxN1-Cre mice. In addition, γδ T cells were absent from the fetal thymus and epidermis of IL-7(f/f) FoxN1-Cre mice. Furthermore, TCRγδ(+) intraepithelial lymphocytes (IELs) were significantly decreased in the small intestines of IL-7(f/f) FoxN1-Cre mice. To evaluate the function of IL-7 produced in the intestine, we crossed the IL-7(f/f) mice with villin promoter-driven Cre (Vil-Cre) mice to obtain the mice deficient in IL-7 production from intestinal epithelial cells. We observed that αß and γδ IELs of IL-7(f/f) Vil-Cre mice were comparable to control mice. Collectively, our results suggest that TEC-derived IL-7 plays a major role in proliferation, survival, and maturation of thymocytes and is indispensable for γδ T cell development. This study also demonstrates that IL-7 produced in the thymus is essential for the development of γδ IELs and indicates the thymic origin of γδ IELs.

MicroRNAs control the maintenance of thymic epithelia and their competence for T lineage commitment and thymocyte selection.

Thymic epithelial cells provide unique cues for the lifelong selection and differentiation of a repertoire of functionally diverse T cells. Rendered microRNA (miRNA) deficient, these stromal cells in the mouse lose their capacity to instruct the commitment of hematopoietic precursors to a T cell fate, to effect thymocyte positive selection, and to achieve promiscuous gene expression required for central tolerance induction. Over time, the microenvironment created by miRNA-deficient thymic epithelia assumes the cellular composition and structure of peripheral lymphoid tissue, where thympoiesis fails to be supported. These findings emphasize a global role for miRNA in the maintenance and function of the thymic epithelial cell scaffold and establish a novel mechanism how these cells control peripheral tissue Ag expression to prompt central immunological tolerance.

The thymic epithelial microRNA network elevates the threshold for infection-associated thymic involution via miR-29a mediated suppression of the IFN-α receptor.

Thymic output is a dynamic process, with high activity at birth punctuated by transient periods of involution during infection. Interferon-α (IFN-α) is a critical molecular mediator of pathogen-induced thymic involution, yet despite the importance of thymic involution, relatively little is known about the molecular integrators that establish sensitivity. Here we found that the microRNA network dependent on the endoribonuclease Dicer, and specifically microRNA miR-29a, was critical for diminishing the sensitivity of the thymic epithelium to simulated infection signals, protecting the thymus against inappropriate involution. In the absence of Dicer or the miR-29a cluster in the thymic epithelium, expression of the IFN-α receptor by the thymic epithelium was higher, which allowed suboptimal signals to trigger rapid loss of thymic cellularity.

Thymic cysts originate from Foxn1 positive thymic medullary epithelium.

Thymic epithelial cells (TECs), derived from polarized two-dimensional (2D) oriented endodermal cells, are distinguished from other epithelial cells by their unique three-dimensional (3D) phenotype. However, some polarized epithelial cells remain present in the normal thymus, forming thymic cysts at the cortico-medullary junction. Here, we analyse the dynamics, origin and phenotype of such thymic cysts. In time-course experiments, we show a reverse correlation between thymic cyst expansion and the presence of thymocytes, suggesting a default pathway for the development of TECs in the absence of thymocytes. By transplanting isolated TEC populations into E15 fetal thymic lobes, we provide evidence that medullary thymic epithelial cells (mTECs), rather than cortical thymic epithelial cells (cTECs) contribute to the formation of thymic cysts. Finally, thymi of reporter mice reveal that the cysts originate from epithelia committed to a thymic fate, as indicated by the expression of Foxn1. The 2D-phenotype of cyst-lining TECs is not caused by a downregulation of Foxn1 expression, since a significant proportion of these cells in the embryonic and adult thymus continues to express Foxn1 at the protein level.

Stabilized beta-catenin in thymic epithelial cells blocks thymus development and function.

Thymic T cell development is dependent on a specialized epithelial microenvironment mainly composed of cortical and medullary thymic epithelial cells (TECs). The molecular programs governing the differentiation and maintenance of TECs remain largely unknown. Wnt signaling is central to the development and maintenance of several organ systems but a specific role of this pathway for thymus organogenesis has not yet been ascertained. In this report, we demonstrate that activation of the canonical Wnt signaling pathway by a stabilizing mutation of beta-catenin targeted exclusively to TECs changes the initial commitment of endodermal epithelia to a thymic cell fate. Consequently, the formation of a correctly composed and organized thymic microenvironment is prevented, thymic immigration of hematopoietic precursors is restricted, and intrathymic T cell differentiation is arrested at a very early developmental stage causing severe immunodeficiency. These results suggest that a precise regulation of canonical Wnt signaling in thymic epithelia is essential for normal thymus development and function.

Delta-like 4 is indispensable in thymic environment specific for T cell development.

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

Maintenance of a normal thymic microenvironment and T-cell homeostasis require Smad4-mediated signaling in thymic epithelial cells.

Signals mediated by the transforming growth factor-beta superfamily of growth factors have been implicated in thymic epithelial cell (TEC) differentiation, homeostasis, and function, but a direct reliance on these signals has not been established. Here we demonstrate that a block in canonical transforming growth factor-beta signaling by the loss of Smad4 expression in TECs leads to qualitative changes in TEC function and a progressively disorganized thymic microenvironment. Moreover, the number of thymus resident early T-lineage progenitors is severely reduced in the absence of Smad4 expression in TECs and directly correlates with extensive thymic and peripheral lymphopenia. Our observations hence place Smad4 within the signaling events in TECs that determine total thymus cellularity by controlling the number of early T-lineage progenitors.