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.
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 α 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.
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.
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.
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.
A novel 3D scaffold derived from the thymic extracellular matrix seeded in vitro by either fetal or adult thymic epithelial cells (TEC) supports thymopoiesis over an extended period of time. The success of engineering a thymic scaffold sustaining TEC function provides unprecedented opportunities to investigate thymus development and physiology ex vivo and to design and implement novel strategies for thymus replacement therapies.
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.
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.
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.
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.
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.
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.
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.
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.
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