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.

Thymic epithelial organoids mediate T cell development.

Although the advent of organoids opened unprecedented perspectives for basic and translational research, immune system-related organoids remain largely underdeveloped. Here we established organoids from the thymus, the lymphoid organ responsible for T cell development. We identified conditions enabling thymic epithelial progenitor cell proliferation and development into organoids with diverse cell populations and transcriptional profiles resembling in vivo thymic epithelial cells (TECs) more closely than traditional TEC cultures. Contrary to these two-dimensional cultures, thymic epithelial organoids maintained thymus functionality in vitro and mediated physiological T cell development upon reaggregation with T cell progenitors. The reaggregates showed in vivo-like epithelial diversity and ability to attract T cell progenitors. Thymic epithelial organoids are the first organoids originating from the stromal compartment of a lymphoid organ. They provide new opportunities to study TEC biology and T cell development in vitro, paving the way for future thymic regeneration strategies in ageing or acute injuries.

Thymus Extracellular Matrix-Derived Scaffolds Support Graft-Resident Thymopoiesis and Long-Term In Vitro Culture of Adult Thymic Epithelial Cells.

The thymus provides the physiological microenvironment critical for the development of T lymphocytes, the cells that orchestrate the adaptive immune system to generate an antigen-specific response. A diverse population of stroma cells provides surface-bound and soluble molecules that orchestrate the intrathymic maturation and selection of developing T cells. Forming an intricate 3D architecture, thymic epithelial cells (TEC) represent the most abundant and important constituent of the thymic stroma. Effective models for in and ex vivo use of adult TEC are still wanting, limiting the engineering of functional thymic organoids and the understanding of the development of a competent immune system. Here a 3D scaffold is developed based on decellularized thymic tissue capable of supporting in vitro and in vivo thymopoiesis by both fetal and adult TEC. For the first time, direct evidences of feasibility for sustained graft-resident T-cell development using adult TEC as input are provided. Moreover, the scaffold supports prolonged in vitro culture of adult TEC, with a retained expression of the master regulator Foxn1. The success of engineering a thymic scaffold that sustains adult TEC function provides unprecedented opportunities to investigate thymus development and physiology and to design and implement novel strategies for thymus replacement therapies.

Violation of the 12/23 rule of genomic V(D)J recombination is common in lymphocytes.

V(D)J genomic recombination joins single gene segments to encode an extensive repertoire of antigen receptor specificities in T and B lymphocytes. This process initiates with double-stranded breaks adjacent to conserved recombination signal sequences that contain either 12- or 23-nucleotide spacer regions. Only recombination between signal sequences with unequal spacers results in productive coding genes, a phenomenon known as the "12/23 rule." Here we present two novel genomic tools that allow the capture and analysis of immune locus rearrangements from whole thymic and splenic tissues using second-generation sequencing. Further, we provide strong evidence that the 12/23 rule of genomic recombination is frequently violated under physiological conditions, resulting in unanticipated hybrid recombinations in ∼10% of Tcra excision circles. Hence, we demonstrate that strict adherence to the 12/23 rule is intrinsic neither to recombination signal sequences nor to the catalytic process of recombination and propose that nonclassical excision circles are liberated during the formation of antigen receptor diversity.

A subpopulation of CD103(pos) ICOS(pos) Treg cells occurs at high frequency in lymphopenic mice and represents a lymph node specific differentiation stage.

Regulatory T (Treg) cells are pivotal for the maintenance of peripheral tolerance by controlling self-reactive, chronic, and homeostatic T-cell responses. Here, we report that the increase in Treg-cell suppressive function observed in lymphopenic mice correlates with the degree of lymphopenia and is caused by a higher frequency of a novel subpopulation of CD103(pos) ICOS(pos) Treg cells. Though present in the thymus, CD103(pos) ICOS(pos) Treg cells are not generated there but recirculate from the periphery to that site. The acquisition and maintenance of this distinctive phenotype requires the LN microenvironment and the in situ availability of antigen. Contrary to conventional effector and other Treg cells, the cellularity of CD103(pos) ICOS(pos) Treg cells is not affected by the absence of IL-7 and thymic stroma lymphopoetin. Given their increased frequency in lymphopenia, the absolute number of CD103(pos) ICOS(pos) Treg cells remains unchanged in the periphery irrespective of a paucity of total Treg cells. We furthermore demonstrate, with cell transfers in mice, that the CD103(pos) ICOS(pos) phenotype represents a LN-specific differentiation stage arrived at by several other Treg-cell subsets. Thus, tissue-specific cues determine the overall potency of the peripheral Treg-cell pool by shaping its subset composition.

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.

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.

T cell regulation as a side effect of homeostasis and competition.

We have previously hypothesized that maintaining a balanced peripheral immune system may not be the sole responsibility of a specialized subset of T cells dedicated to immune regulation, but also a side effect of normal competition for shared resources within an intact immune system. Here we show that regulatory activity is correlated with high homeostatic expansion potential, reflecting the avidity for self-peptide:MHC complexes. Monoclonal transgenic T cells with high homeostatic expansion potential and lacking characteristics previously associated with regulatory function were able to regulate wasting disease induced by transfer of a small number of naive CD45RB(hi) CD4 T cells into lymphopenic hosts. Self-regulatory function is also found in the naive polyclonal T cell repertoire depleted of CD25(+) T cells. T cells capable of preventing immune pathology, like the transgenic T cells, express higher than average levels of CD5, an indicator of avidity for self:MHC peptide complexes. We therefore propose that dysregulated expansion of potentially pathogenic T cells in a lymphopenic environment can be prevented by members of the naive T cell repertoire, irrespective of their specificity, as a side effect of their response to homeostatic and antigenic stimulation.

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.

TGF-beta signaling in thymic epithelial cells regulates thymic involution and postirradiation reconstitution.

The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in reduced thymopoietic capacity have been suggested to account for this physiologic process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-beta signaling in thymic epithelial cells exerts a direct influence on the cell's capacity to support thymopoiesis in the aged mouse as the physiologic process of thymic senescence is mitigated in mice deficient for the expression of TGF-beta RII on thymic epithelial cells. Moreover, TGF-beta signaling in these stromal cells transiently hinders the early phase of thymic reconstitution after myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-beta signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis after hematopoietic stem cell transplantation.

A short primer on early molecular and cellular events in thymus organogenesis and replacement.

Haematopoietic precursors have to undergo a complex series of maturational steps in the thymus before they exit into the periphery as functional T lymphocytes. Thymic stroma cells, the majority being of epithelial origin, provide the functional partners for the maturational progression along this differentiation pathway. Here we review some of the molecular and cellular mechanisms that account for thymus organogenesis and discuss a strategy to use thymic epithelial precursor cells for the regeneration of the thymic microenvironment.

Differential Response of Mouse Thymic Epithelial Cell Types to Ionizing Radiation-Induced DNA Damage.

Thymic epithelial cells (TECs) are the main components of the thymic stroma that support and control T-cell development. Preparative regimens using DNA-damaging agents, such as total body irradiation and/or chemotherapeutic drugs, that are necessary prior to bone marrow transplantation (BMT) have profound deleterious effects on the hematopoietic system, including the thymic stroma, which may be one of the main causes for the prolonged periods of T-cell deficiency and the inefficient T cell reconstitution that are common following BMT. The DNA damage response (DDR) is a complex signaling network that allows cells to respond to all sorts of genotoxic insults. Hypoxia is known to modulate the DDR and play a role affecting the survival capacity of different cell types. In this study, we have characterized in detail the DDR of cortical and medullary TEC lines and their response to ionizing radiation, as well as the effects of hypoxia on their DDR. Although both mTECs and cTECs display relatively high radio-resistance, mTEC cells have an increased survival capacity to ionizing radiation (IR)-induced DNA damage, and hypoxia specifically decreases the radio-resistance of mTECs by upregulating the expression of the pro-apoptotic factor Bim. Analysis of the expression of TEC functional factors by primary mouse TECs showed a marked decrease of highly important genes for TEC function and confirmed cTECs as the most affected cell type by IR. These findings have important implications for improving the outcomes of BMT and promoting successful T cell reconstitution.

A short primer on early molecular and cellular events in thymus organogenesis and replacement.

Haematopoietic precursors have to undergo a complex series of maturational steps in the thymus before they exit into the periphery as functional T lymphocytes. Thymic stroma cells, the majority being of epithelial origin, provide the functional partners for the maturational progression along this differentiation pathway. Here we review some of the molecular and cellular mechanisms that account for thymus organogenesis and discuss a strategy to use thymic epithelial precursor cells for the regeneration of the thymic microenvironment.

Cell Growth Dynamics in Embryonic and Adult Mouse Thyroid Revealed by a Novel Approach to Detect Thyroid Gland Subpopulations.

BACKGROUND: The thyroid is composed of endocrine epithelial cells, blood vessels, and mesenchyme. However, no data exist thus far on absolute cell numbers, relative distribution, and proliferation of the different cell populations in the developing and mature thyroid. The aim of this study was therefore to establish a flow cytometry protocol that allows detection and quantification of discrete cell populations in embryonic and adult murine thyroid tissues. METHODS: Cell-type anti-mouse specific antibodies were used for erythroid cells (Ter119), hematopoietic cells (CD45), epithelial cells (EpCam/CD326, E-cadherin/CD324), thyroid follicular cells and C-cells (Nkx2-1), endothelial cells (Pecam/CD31, Icam-1/CD54), and fibroblasts (PDGFRa/CD140a). Proliferating cells were detected after labeling with 5-bromo-2'-deoxyuridine (BrdU). For flow cytometry analyses, micro-dissected embryonic (E) and adult thyroids were pooled (E13.5, n = 25; E15.5, n = 15; E17.5, n = 15; adult, n = 4) in one sample. RESULTS: The absolute parenchymal cell numbers per mouse thyroid (M ± SD), excluding the large number of CD45(+) and Ter119(+) cells, increased from 7425 ± 1338 at E13.5 to 271,561 ± 22,325 in adult tissues. As expected, Nkx2-1(+) cells represented the largest cell population in adult tissues (61.2 ± 1.1%). Surprisingly, at all three embryonic stages analyzed, thyroid follicular cells and C-cells accounted only for a small percentage of the total thyroid cell mass (between 4.7 ± 0.4% and 9.4 ± 1.6%). In contrast, the largest cell population at all three embryonic stages was identified as PDGFRa/CD140a(+) fibroblasts (61.4 ± 0.4% to 77.3 ± 1.1%). However, these cells represented the smallest population in adult tissues (5.2 ± 0.8%). Pecam/CD31(+) endothelial cells increased from E13.5 to E15.5 from 3.7 ± 0.8% to 8.5 ± 3.0%, then remained stable at E17.5 and adult tissues. Proliferation rates were sizable during the entire organogenesis but differed between cell populations, with distinct proliferative peaks at E13.5 in epithelial cells (32.7 ± 0.6% BrdU(+) cells), and at E15.5 in endothelial cells (22.4 ± 2.4% BrdU(+) cells). Fibroblasts showed a constant proliferation rate in embryonic tissues. In adult tissues, BrdU(+) cells were between 0.1% and 0.4% in all cell types. CONCLUSIONS: Using a novel flow cytometry-based method, a previously unobserved highly dynamic growth pattern of thyroid cell populations during embryogenesis was uncovered. This approach will provide a useful new tool for cell function analyses in murine thyroid disease models.

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility.

The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell-specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.

EVA regulates thymic stromal organisation and early thymocyte development.

Epithelial V-like antigen (EVA) is an immunoglobulin-like adhesion molecule identified in a screen for molecules developmentally regulated at the DN to DP progression in thymocyte development. We show that EVA is expressed during the early stages of thymus organogenesis in both fetal thymic epithelia and T cell precursors, and is progressively downregulated from day 16.5 of embryonic development. In the postnatal thymus, EVA expression is restricted to epithelial cells and is distributed throughout both cortical and medullary thymic regions. Transgenic overexpression of EVA in the thymus cortex resulted in a modified stromal environment, which elicited an increase in organ size and absolute cell number. Although peripheral T lymphocyte numbers are augmented throughout life, no imbalance either in the repertoire, or in the different T cell subsets was detected. Collectively, these data suggest a role for EVA in structural organisation of the thymus and early lymphocyte development.

CD25+ CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production.

Maintenance of homeostasis in the immune system involves competition for resources between T lymphocytes, which avoids the development of immune pathology seen in lymphopenic mice. CD25+ CD4+ T cells are important for homeostasis, but there is as yet no consensus on their mechanisms of action. Although CD25+ CD4+ T cells cause substantial down-regulation of IL-2 mRNA in responder T cells in an in vitro co-culture system, the presence of IL-protein can be demonstrated by intracellular staining. As a consequence of competition for IL-2, CD25+ CD4+ T cells further up-regulate the IL-2R alpha chain (CD25), a process that is strictly dependent on IL-2, whereas responder T cells fail to up-regulate CD25. Similarly, adoptive transfer into lymphopenic mice showed that CD25+ CD4+ T cells interfere with CD25 up-regulation on co-transferred naive T cells, while increasing their own CD25 levels. IL-2 sequestration by CD25+ CD4+ T cells is not a passive phenomenon but instead initiates--in conjunction with signals through the TCR--their differentiation to IL-10 production. Although IL-10 is not required for in vitro suppression, it is vital for the in vivo function of regulatory T cells. Our data provide a link explaining the apparent difference in regulatory mechanisms in vitro and in vivo.

Factors affecting the susceptibility of the mouse pituitary gland to CD8 T-cell-mediated autoimmunity.

We have previously shown, in a transgenic mouse model, that the pituitary gland is susceptible to CD8 T-cell-mediated autoimmunity, triggered by a cell-specific model autoantigen, resulting in pan-anterior pituitary hypophysitis and dwarfism. In the present study, we now demonstrate that antigen dose, the T-cell precursor frequency, the degree of lymphopenia and the context of target antigen expression, are important parameters determining the time course and extent of the pathological consequences of CD8 T-cell-mediated autoimmunity. Furthermore, our data indicate that the pituitary gland is susceptible to CD8 autoimmunity following an inflammatory insult such as a viral infection. As lymphocytic hypophysitis may be manifest in other autoimmune conditions, and the pituitary gland may be susceptible to T-cell-mediated pathology after immunization with a virus expressing soluble pituitary antigen, it is important to consider that strategies based on vaccination against soluble pituitary gonadotrophins could have other unexpected endocrine consequences.

Activation of CD8 T cells by antigen expressed in the pituitary gland.

Ag expressed exclusively in the anterior pituitary gland and secreted locally by pituitary somatotrophs can gain access to the MHC class I presentation pathway and activate CD8 T cells. Influenza nucleoprotein (NP) was expressed as a transgene under the control of the human growth hormone (GH) locus control region. Activation of monoclonal F5 CD8 T cells specific for NP resulted in spontaneous autoimmune pathology of the pituitary gland in mice transgenic for both NP and the F5 TCR. Destruction of somatotrophs resulted in drastically reduced GH levels in adult mice and a dwarf phenotype. Adoptive transfer of F5 T cells into NP-transgenic hosts resulted in full T cell activation, first demonstrable in regional lymph nodes, followed by their migration to the pituitary gland. Despite the presence of activated, IFN-gamma-producing CD8 T cells in the pituitary gland and a slight reduction in pituitary GH levels, no effect on growth was observed. Thus, CD8 T cells have access to the neuroendocrine system and get fully activated in the absence of CD4 help, but Ag recognition in this location causes autoimmune pathology only in the presence of excessive CD8 T cell numbers.

IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells.

Regulatory T cells (T(Reg)) control immune responses to self and nonself Ags. The relationship between Ag-driven IL-10-secreting T(Reg) (IL-10-T(Reg)) and naturally occurring CD4(+)CD25(+) T(Reg) is as yet unclear. We show that mouse IL-10-T(Reg) obtained using either in vitro or in vivo regimens of antigenic stimulation did not express the CD4(+)CD25(+) T(Reg)-associated transcription factor Foxp3. However, despite the absence of Foxp3 expression, homogeneous populations of IL-10-T(Reg) inhibited the in vitro proliferation of CD4(+)CD25(-) T cells with a similar efficiency to that of CD4(+)CD25(+) T(Reg). This inhibition of T cell proliferation by IL-10-T(Reg) was achieved through an IL-10-independent mechanism as seen for CD4(+)CD25(+) T(Reg) and was overcome by exogenous IL-2. Both IL-10-T(Reg) and CD4(+)CD25(+) T(Reg) were similar in that they produced little to no IL-2. These data show that Foxp3 expression is not a prerequisite for IL-10-T(Reg) activity in vitro or in vivo, and suggest that IL-10-T(Reg) and naturally occurring CD4(+)CD25(+) T(Reg) may have distinct origins.