Essential role of IkappaB kinase alpha in thymic organogenesis required for the establishment of self-tolerance

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.     

Essential role of NF-kappa B-inducing kinase in T cell activation through the TCR/CD3 pathway

NF-kappa B-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-beta receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-kappa B activity in both mature and immature T cells; the impaired NF-kappa B activity in mature T cells was also associated with the failure of maintenance of activated NF-kappa B. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-kappa B activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.     

NF kappa B-inducing kinase deficiency results in the development of a subset of regulatory T cells, which shows a hyperproliferative activity upon glucocorticoid-induced TNF receptor family-related gene stimulation

CD4+CD25+ regulatory T cells (T(reg)) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on T(reg) has been shown to be a key player of regulating T(reg)-mediated suppression. A recent study reports that NF-kappaB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of T(reg) but not for its suppression capacity. In this report, we have shown that T(reg) from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive T(reg) proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type T(reg). Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62L(low) T(reg) both in thymus and periphery than their wild-type littermates. This CD62(low) subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.     

BMP signaling is required for normal thymus development

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.     

T cell development critically depends on prethymic stromal patched expression

We recently described that T cell specification in mice deficient in the Hedgehog (Hh) receptor Patched (Ptch) is blocked at the level of the common lymphoid progenitor in the bone marrow (BM). Adoptive transfer of wild-type BM in Ptch-deficient mice provides evidence that T cell development strictly depends on Ptch expression in the nonhematopoietic compartment. Transplantation experiments using BM deficient in the glucocorticoid receptor exclude any involvement of the stress hormone corticosterone in our model. Using cell-type-specific knockout mice, we show that T cell development is independent of T cell-intrinsic Ptch expression. Furthermore, Ptch expression by the thymus stroma is dispensable, as revealed by fetal thymus organ culture and thymus transplantation. In contrast, analysis of the earliest thymic progenitors in Ptch-deficient mice indicated that Ptch is required for the development or supply of thymic homing progenitors that give rise to earliest thymic progenitors. Collectively, our findings identified Ptch as an exclusive T cell-extrinsic factor necessary for proper development of T cells at their prethymic stage. This observation may be important for current considerations using Hh inhibitors upstream of Ptch in diseases accompanied by aberrant Hh signaling.     

IL-10 transgenic mice present a defect in T cell development reminiscent of SCID patients.

To analyze the effect of IL-10 overexpressed by APCs as observed in some SCID patients, we have expressed the human IL-10 cDNA under the control of the murine MHC class II promoter in transgenic mice. Similar to SCID patients, these mice presented a defect in T cell maturation characterized by a rapid thymic aplasia that started after birth. The blockage in T cell maturation was strictly restricted to TCR-alpha beta T cells as the absolute number of thymic dendritic, TCR-gamma delta and NK1.1 T cells were equivalent to control littermates. Crossing IL-10 transgenic mice with TCR transgenic mice or treatment with staphylococcal enterotoxin B showed that the defect was not related to the impairment of positive or negative selection. However, repopulating of IL-10 transgenic mouse-fetal thymic organ culture with different stages of triple negative T cells isolated from control mice showed that the blockage occurred specifically at the pre-T cell stage and was reverted by treatment with blocking anti-IL-10 mAbs. These results demonstrate that IL-10 regulates T cell maturation and that dysregulation of IL-10 expression can lead to severe T cell immunodeficiency.     

NF-kappaB2 is required for the control of autoimmunity by regulating the development of medullary thymic epithelial cells

Medullary thymic epithelial cells function as antigen-presenting cells in negative selection of self-reactive T cell clones, a process essential for the establishment of central self-tolerance. These cells mirror peripheral tissues through promiscuous expression of a diverse set of tissue-restricted self-antigens. The genes and signaling pathways that regulate the development of medullary thymic epithelial cells are not fully understood. Here we show that mice deficient in NF-kappaB2, a member of the NF-kappaB family, display a marked reduction in the number of mature medullary thymic epithelial cells that express CD80 and bind the lectin Ulex europaeus agglutinin-1, leading to a significant decrease in the extent of promiscuous gene expression in the thymus of NF-kappaB2(-/-) mice. Moreover, NF-kappaB2(-/-) mice manifest autoimmunity characterized by multiorgan infiltration of activated T cells and high levels of autoantibodies to multiple organs. A subpopulation of the mice also develops immune complex glomerulonephritis. These findings identify a physiological function of NF-kappaB2 in the development of medullary thymic epithelial cells and, thus, the control of self-tolerance induction.     

Abnormal immune function of hemopoietic cells from alymphoplasia (aly) mice, a natural strain with mutant NF-kappa B-inducing kinase

Alymphoplasia (aly) mice, a natural strain with a mutant NF-kappa B-inducing kinase (NIK) gene, manifest a unique phenotype; they lack lymph nodes and Peyer's patches, have a disturbed spleen architecture, and exhibit defects in both Ab and cellular immune responses. Although a stromal defect caused by impaired lymphotoxin-beta receptor signaling accounts for their abnormal lymphoid organogenesis, the exact mechanisms underlying the development of immunodeficiency in aly mice are poorly understood. We therefore investigated the contribution of hemopoietic cells with the aly NIK mutation to the development of immunodeficiency. Transfer of aly/aly bone marrow cells into aly/+ mice resulted in poorly developed B cell follicles and lack of support for the development of germinal centers and isotype switching, indicating that the hemopoietic cells of aly mice contain an autonomous defect. However, follicular dendritic cell clusters were maintained in the spleens of these bone marrow chimeras, suggesting that the lack of follicular dendritic cell clusters in aly mice is probably due to the stromal defect. The aly mice lacked marginal zone B cells in their spleens, and aly/aly B cells showed an impaired proliferative response after in vitro stimulation. IL-2 production by activated T cells was also impaired. By contrast, the dendritic cells of aly mice exhibited grossly normal development and function. Supporting the concept of an autonomous cell defect, Rel protein expression was altered in aly/aly spleens. Thus, the aly NIK mutation affects hemopoietic cell function in an intrinsic fashion and, together with the stromal defect, may contribute to the development of immunodeficiency in aly mice.     

Label Retention Identifies a Multipotent Mesenchymal Stem Cell-Like Population in the Postnatal Thymus

Thymic microenvironments are essential for the proper development and selection of T cells critical for a functional and self-tolerant adaptive immune response. While significant turnover occurs, it is unclear whether populations of adult stem cells contribute to the maintenance of postnatal thymic epithelial microenvironments. Here, the slow cycling characteristic of stem cells and their property of label-retention were used to identify a K5-expressing thymic stromal cell population capable of generating clonal cell lines that retain the capacity to differentiate into a number of mesenchymal lineages including adipocytes, chondrocytes and osteoblasts suggesting a mesenchymal stem cell-like phenotype. Using cell surface analysis both culture expanded LRCs and clonal thymic mesenchymal cell lines were found to express Sca1, PDGFRα, PDGFRβ,CD29, CD44, CD49F, and CD90 similar to MSCs. Sorted GFP-expressing stroma, that give rise to TMSC lines, contribute to thymic architecture when reaggregated with fetal stroma and transplanted under the kidney capsule of nude mice. Together these results show that the postnatal thymus contains a population of mesenchymal stem cells that can be maintained in culture and suggests they may contribute to the maintenance of functional thymic microenvironments.     

Cutting edge: perforin down-regulates CD4 and CD8 T cell-mediated immune responses to a transplanted organ

Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.     

Diabetes resistance/susceptibility in T cells of nonobese diabetic mice conferred by MHC and MHC-linked genes

Polymorphism of MHC and MHC-linked genes is tightly associated with susceptibility to type 1 diabetes (T1D) in human and animal models. Despite the extensive studies, however, the role of MHC and MHC-linked genes expressed by T cells on T1D susceptibility remains unclear. Because T cells develop from TCR(-) thymic precursor (pre-T) cells that undergo MHC restriction mediated by thymic stroma cells, we reconstituted the T cell compartment of NOD.scid-RIP-B7.1 mice using pre-T cells isolated from NOD, NOR, AKR, and C57BL/6 (B6) mice. T1D developed rapidly in the mice reconstituted with pre-T cells derived from NOD or NOR donors. In contrast, most of the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from AKR or B6 donors were free of T1D. Further analysis revealed that genes within MHC locus of AKR or B6 origin reduced incidence of T1D in the reconstituted NOD.scid-RIP-B7.1 mice. The expression of MHC class I genes of k, but not b haplotype, in T cells conferred T1D resistance. Replacement of an interval near the distal end of the D region in T cells of B6 origin with an identical allele of 129.S6 origin resulted in T1D development in the reconstituted mice. These results provide evidence that the expression of MHC class I and MHC-linked genes in T cells of NOD mice indeed contributes to T1D susceptibility, while expression of specific resistance alleles of MHC or MHC-linked genes in T cells alone would effectively reduce or even prevent T1D.     

Regulatory phenomena in tolerance induction

The implication of regulatory T cells in self-tolerance induction was shown in experimental models based on construction of thymic chimera and peripheric T cell transfers. The role played by the epithelial stroma of the thymus in CD4+ regulatory T cell selection was demonstrated. In the NOD (Non Obese Diabetic) strain, protection again diabetes was obtained by grafting supplementary thymuses injected with allogeneic pancreatic islets. This result suggest that the NOD thymuses are defective in the production of T regulatory cells.     

The myelopoietic inducing potential of mouse thymic stromal cells

The thymus has generally been considered as being solely involved in T cell maturation. In this study we have demonstrated that mouse thymic stroma can also support myelopoiesis. Bone marrow from mice treated with 5-fluorouracil was depleted of cells expressing Mac-1, CD4, and CD8 and incubated on lymphocyte-free monolayer cultures of adherent thymic stromal cells. After 7 days there was a marked increase in nonadherent cells, the majority of which were Mac-1+, FcR+, and HSA+. These proliferating bone marrow cells also expressed markers (MTS 17 and MTS 37) found on thymic stromal cells. Such cells were not found in thymic cultures alone, in bone marrow cultured alone, or on control adherent cell monolayers. Supernatants from the cultured thymic stroma, however, were able to induce these cell types in the bone marrow precursor population. Incubation of normal thymocytes with a monolayer of these in vitro cultivated Mac-1+, MTS 17+, MTS 37+ myeloid cells leads to selective phagocytosis of CD4+ CD8+ cells. Hence, this study demonstrates that the thymic adherent cells can induce myelopoiesis in bone marrow-derived precursor cells and provide a form of self-renewal for at least one population of thymic stromal cells. Furthermore, these induced cells are capable of selective phagocytosis of CD4+ CD8+ thymocytes and may provide one mechanism for the selective removal of such cells from the thymus.     

Rac1 deletion causes thymic atrophy

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.     

Regulatory T cells in the establishment and maintenance of self-tolerance: role of the thymic epithelium

The thymus constitutes the microenvironment for T lymphocyte differentiation and acquisition of self-tolerance. Aiming to specify the contributions of the two essential parts of the thymus, namely hemopoietic and epithelial, we have devised experimental models in birds and mice. Chimeric thymuses, xenogeneic in birds and allogeneic in mice, were constructed early in development. In both models we could demonstrate a critical role of the epithelial component of the thymic stroma in induction and maintenance of self-tolerance. These experiments showed that suppression mechanisms are also implicated in these events, strongly suggesting the existence of regulatory T cells in both models. Before these experiments the control of self-tolerance was usually attributed to suppressive cells. However, as the cell phenotypes were not identified, the role of these cells was disregarded. Numerous studies since our investigations argue in favour of regulatory mechanisms. The work we initiated several years ago represents a contribution to our understanding of the two linked and opposite aspects of immune-responded control, namely self-tolerance and autoimmunity.     

Molecular signature of recent thymic selection events on effector and regulatory CD4+ T lymphocytes

Natural CD4+CD25+ regulatory T lymphocytes (Treg) are key protagonists in the induction and maintenance of peripheral T cell tolerance. Their thymic origin and biased repertoire continue to raise important questions about the signals that mediate their development. We validated analysis of MHC class II capture by developing thymocytes from thymic stroma as a tool to study quantitative and qualitative aspects of the cellular interactions involved in thymic T cell development and used it to analyze Treg differentiation in wild-type mice. Our data indicate that APCs of bone marrow origin, but, surprisingly and importantly, not thymic epithelial cells, induce significant negative selection among the very autoreactive Treg precursors. This fundamental difference between thymic development of regulatory and effector T lymphocytes leads to the development of a Treg repertoire enriched in cells specific for a selected subpopulation of self-Ags, i.e., those specifically expressed by thymic epithelial cells.     

Reduced interleukin-1 responsiveness in immune system and central nervous system of inbred polydipsic (STR/N) mice

Inbred polydipsic mice (STR/N strain) have primary polydipsia. The previous studies found abnormalities in the central nervous system (CNS), especially in the hypothalamus and circumventricular organ. As a part of pursuing to find the cause of the polydipsia, we investigated immunological characteristics of STR/N mice, using the ICR strain of mice as control. Their thymic subset cells showed that CD4+CD8+ double positive cells were increased, CD4+ single positive cells were decreased and CD5 expression was deficient, compared to ICR mice. T cell proliferative response and interleukin (IL)-2 production caused by IL-1beta stimulation were reduced in STR/N mice than those in the ICR mice. In in vivo studies the degree of thymic atrophy and the increases in serum level of ACTH and corticosterone induced by intraperitoneal IL-1beta injection were much less in STR/N mice than those in controls. Furthermore, adipsic response also induced by IL-1beta injection was greatly reduced compared to their control mice. All these results suggest that the responsiveness to IL-1 is impaired both in the immune system and the CNS of STR/N mice.     

Dioxin-induced adseverin expression in the mouse thymus is strictly regulated and dependent on the aryl hydrocarbon receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ligand for the ubiquitous, intracellular aryl hydrocarbon receptor (AhR), up-regulates the actin-modulating protein adseverin in mouse lymphoid tissues, a response that may be correlated to the immunotoxicity of TCDD. Here, by using chimeric mice with TCDD-responsive (AhR(+/+)) hematopoietic cells and TCDD-unresponsive (AhR(minus sign/minus sign)) thymic stroma, or the reverse, we show that TCDD-induced expression of adseverin in thymus is dependent on AhR expression in hematopoietic cells but not in stroma. The use of fetal thymic organ cultures also indicates that TCDD-induced expression of adseverin is confined to the thymocytes. The thymic stroma showed no induction of adseverin expression after TCDD exposure, although TCDD clearly activated the AhR in these cells, as indicated by the induction of CYP1A1. Adseverin was not induced in the thymus of normal adult C57BL/6 mice exposed to beta-estradiol or dexamethasone, two other agents, which also cause thymic atrophy. This further supports that adseverin induction is a specific gene regulatory effect by TCDD on thymocytes.