Interferon-γ resistance and immune evasion in glioma develop via Notch-regulated co-evolution of malignant and immune cells.

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of T cells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled by upregulation of oncogenes and downregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control.

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.

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.

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.

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.

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.

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.

Normal erythropoiesis but severe polyposis and bleeding anemia in Smad4-deficient mice.

The tumor suppressor Smad4 mediates signaling by the transforming growth factor beta (TGF-beta) superfamily of ligands. Previous studies showed that several TGF-beta family members exert important functions in hematopoiesis. Here, we studied the role of Smad4 in adult murine hematopoiesis using the inducible Mx-Cre/loxP system. Mice with homozygous Smad4 deletion (Smad4(Delta/Delta)) developed severe anemia 6 to 8 weeks after induction (mean hemoglobin level 70 g/L). The anemia was not transplantable, as wild-type mice reconstituted with Smad4(Delta/Delta) bone marrow cells had normal peripheral blood counts. These mice did not develop an inflammatory disease typical for mice deficient in TGF-beta receptors I and II, suggesting that the suppression of inflammation by TGF-beta is Smad4 independent. The same results were obtained when Smad4 alleles were deleted selectively in hematopoietic cells using the VavCre transgenic mice. In contrast, lethally irradiated Smad4(Delta/Delta) mice that received wild-type bone marrow cells developed anemia similar to Smad4(Delta/Delta) mice that did not receive a transplant. Liver iron stores were decreased and blood was present in stool, indicating that the anemia was due to blood loss. Multiple polyps in stomach and colon represent a likely source of the bleeding. We conclude that Smad4 is not required for adult erythropoiesis and that anemia is solely the consequence of blood loss.

Donor T-cell alloreactivity against host thymic epithelium limits T-cell development after bone marrow transplantation.

Acute graft-versus-host disease (aGVHD) impairs thymus-dependent T-cell regeneration in recipients of allogeneic bone marrow transplants through yet to be defined mechanisms. Here, we demonstrate in mice that MHC-mismatched donor T cells home into the thymus of unconditioned recipients. There, activated donor T cells secrete IFN-gamma, which in turn stimulates the programmed cell death of thymic epithelial cells (TECs). Because TECs themselves are competent and sufficient to prime naive allospecific T cells and to elicit their effector function, the elimination of host-type professional antigen-presenting cells (APCs) does not prevent donor T-cell activation and TEC apoptosis, thus precluding normal thymopoiesis in transplant recipients. Hence, strategies that protect TECs may be necessary to improve immune reconstitution following allogeneic bone marrow transplantation.