Development of functional thymic epithelial cells occurs independently of lymphostromal interactions

The thymus provides a specialised microenvironment for the development of T-cell precursors. This developmental programme depends upon interactions with stromal cells such as thymic epithelial cells, which provide signals for proliferation, survival and differentiation. In turn, it has been proposed that development of thymic epithelial cells themselves is regulated by signals produced by developing thymocytes. Evidence in support of this symbiotic relationship, termed thymic crosstalk, comes from studies analysing the thymus of adult mice harbouring blocks at specific stages of thymocyte development, where it is difficult to separate mechanisms regulating the initial development of thymic epithelial cells from those regulating their maintenance. To distinguish between these processes, we have analysed the initial developmental programme of thymic epithelial cells within the embryonic thymus, in either the presence or absence of normal T-cell development. We show that keratin 5+8+ precursor epithelial cells present in the early thymic rudiment differentiate into discrete cortical and medullary epithelial subsets displaying normal gene expression profiles, and acquire functional competence, independently of signals from T-cell precursors. Thus, our findings redefine current models of thymus development and argue against a role for thymocyte-epithelial cell crosstalk in the development of thymic epithelial progenitors.     

T cell development from kit-negative progenitors in the Foxn1Delta/Delta mutant thymus

Foxn1Delta is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1Delta/Delta adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1-/- mice were able to differentiate to the DN2 or DN3 stage in the Foxn1Delta/Delta thymus. Our data suggest that thymopoiesis in the Foxn1Delta/Delta adult thymus proceeds from CD117- atypical progenitors, while CD117+ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1Delta/Delta adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.     

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.     

Two developmentally distinct populations of dendritic cells inhabit the adult mouse thymus: demonstration by differential importation of hematogenous precursors under steady state conditions

Although a variety of lymphoid and myeloid precursors can generate thymic dendritic cells (DCs) under defined experimental conditions, the developmental origin(s) of DCs in the steady state thymus is unknown. Having previously used selective combinations of normal, parabiotic, and radioablated mice to demonstrate that blood-borne prothymocytes are imported in a gated and competitive manner, we used a similar approach in this study to investigate the importation of the hematogenous precursors of thymic DCs. The results indicate that two developmentally distinct populations of DC precursors normally enter the adult mouse thymus. The first population is indistinguishable from prothymocytes according to the following criteria: 1) inefficient (<20%) exchange between parabiotic partners; 2) gated importation by the thymus; 3) competitive antagonism for intrathymic niches; 4) temporally linked generation of thymocytes and CD8alpha(high) DCs; and 5) absence from prothymocyte-poor blood samples. The second population differs diametrically from prothymocytes in each of these properties, and appears to enter the thymus in at least a partially differentiated state. The resulting population of DCs has a CD8alpha(-/low) phenotype, and constitutes approximately 50% of total thymic DCs. The presence of two discrete populations of DCs in the steady state thymus implies functional heterogeneity consistent with evidence implicating lymphoid DCs in the negative selection of effector thymocytes and myeloid DCs in the positive selection of regulatory thymocytes.     

Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells

We have recently demonstrated the presence of three populations of dendritic cells (DC) in the murine Peyer's patch. CD11b(+)/CD8alpha(-) (myeloid) DCs are localized in the subepithelial dome, CD11b(-)/CD8alpha(+) (lymphoid) DCs in the interfollicular regions, and CD11b(-)/CD8alpha(-) (double-negative; DN) DCs at both sites. We now describe the presence of a novel population of intraepithelial DN DCs within the follicle-associated epithelium and demonstrate a predominance of DN DCs only in mucosal lymphoid tissues. Furthermore, we demonstrate that all DC subpopulations maintain their surface phenotype upon maturation in vitro, and secrete a distinct pattern of cytokines upon exposure to T cell and microbial stimuli. Only myeloid DCs from the PP produce high levels of IL-10 upon stimulation with soluble CD40 ligand(-) trimer, or Staphylococcus aureus and IFN-gamma. In contrast, lymphoid and DN, but not myeloid DCs, produce IL-12p70 following microbial stimulation, whereas no DC subset produces IL-12p70 in response to CD40 ligand trimer. Finally, we show that myeloid DCs from the PP are particularly capable of priming naive T cells to secrete high levels of IL-4 and IL-10, when compared with those from nonmucosal sites, while lymphoid and DN DCs from all tissues prime for IFN-gamma production. These findings thus suggest that DC subsets within mucosal tissues have unique immune inductive capacities.     

Thymic epithelial cells responsible for impaired generation of NK-T thymocytes in Alymphoplasia mutant mice

We have previously shown that the generation of an NK1.1+TCRalphabeta+ (NK-T) cell population is severely impaired in an alymphoplasia mutant (aly/aly) mouse strain and the defect resides in the thymic environment. In the present study, to elucidate the thymic stromal component(s) that affects the development of NK-T cells, radiation bone marrow chimeras were established with the aly/aly mouse as a donor and either the beta2 microglobulin knockout (beta2m-/-) or the CD1d1-/- mouse that also lacks the NK-T cell population as a recipient. A normal population of NK-T cells with a typical NK-T phenotype and functions was detected in both the thymus and the spleen of these chimeras. These findings indicated that a radiation-resistant CD1(-) component of the thymus supported generation of functional NK-T cells from aly/aly precursors. Furthermore, transfer of an intact medullary thymic epithelial cell line into aly/aly thymus significantly induced the generation of NK-T cells in the thymus. These findings suggest that CD1 molecules of bone marrow-derived cells and the medullary epithelial cells acted in concert in the generation of the NK-T cell population and that a function(s) of the medullary thymic epithelial cells other than direct presentation of CD1 molecules to the NK-T precursors is indispensable for the development of NK-T cells.     

Expression of Notch receptors and ligands on immature and mature T cells

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation.     

Dendritic cell populations in colon and mesenteric lymph nodes of patients with Crohn's disease.

Dendritic cells (DCs) are key cells in innate and adaptive immune responses that determine the pathophysiology of Crohn's disease. Intestinal DCs migrate from the mucosa into mesenteric lymph nodes (MLNs). A number of different markers are described to define the DC populations. In this study we have identified the phenotype and localization of intestinal and MLN DCs in patients with Crohn's disease and non-IBD patients based on these markers. We used immunohistochemistry to demonstrate that all markers (S-100, CD83, DC-SIGN, BDCA1-4, and CD1a) showed a different staining pattern varying from localization in T-cell areas of lymph follicles around blood vessels or single cells in the lamina propria and in the MLN in the medullary cords and in the subcapsular sinuses around blood vessels and in the T-cell areas. In conclusion, all different DC markers give variable staining patterns so there is no marker for the DC.     

Expression of interleukin-1 receptors in the later period of foetal thymic organ culture and during suspension culture of thymocytes from aged mice

Interleukin-1 has been reported to be involved in thymocyte development by exerting a variety of effects on immature CD4-CD8- double-negative (DN) thymocytes. In contrast to the well-documented involvement of IL-1 in thymocyte development, expression of IL-1 receptors (IL-1R) on thymocytes has not been well demonstrated. In the present study, expression of IL-1R on the developing thymocytes was investigated. Although normal thymocytes barely express IL-1R, expression of IL-1R (type I) substantially increased at days 12-15 of foetal thymic organ culture (FTOC), with an increase of the DN subset. The CD4/CD8 profile of the IL-1R (type I)+ cells showed that these cells were mostly restricted to the DN and CD4+CD8+ subsets. Interestingly, in vitro culture of the thymocytes from an aged mouse, but not those from young adult or newborn mice, revealed similar results to those of FTOC. In addition, half of the IL-1R+ cells that increased in the later period of FTOC were gammadelta thymocytes. These results demonstrate IL-1R expression on thymocytes during ex vivo culture and suggest that IL-1R is expressed in a certain environment during normal thymocyte differentiation.     

The thymic insulin-like growth factor axis: involvement in physiology and disease.

A repertoire of neuroendocrine-related genes is transcribed in the non-lymphoid compartment of the thymus, transposing the dual physiological role of this organ at the molecular level in T-cell development towards the establishment of central T-cell self-tolerance. The "neuroendocrine self" has been defined as a series of antigen sequences processed from precursors predominantly expressed in the thymus and first encountered by differentiating T-lymphocytes in their early life. All the members of the insulin gene family are expressed in the thymus according to a precise hierarchy and cellular topography, whereby IGF-II (epithelium of the subcapsular cortex and medulla) exceeds IGF-I (macrophages), which in turn far exceeds INS (rare subsets of medullary epithelial cells). This hierarchy in the degree of their respective thymic expression explains why IGF-II is more tolerated than IGF-I, and much more so than insulin. Evidence has been found for significant regulatory/tolerogenic properties in the IGF-II B:11 - 25 sequence after analysis of the cytokine secretion profile in peripheral blood mononuclear cells isolated from ten DQ8+ type 1 diabetic adolescents. In the thymus, IGF ligands and receptors also intervene in the control of T-cell proliferation and differentiation. Here, we also discuss how a disturbance in the intrathymic IGF-mediated signaling could contribute to the pathogenesis of T-cell leukemia.     

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.     

Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development

The three-dimensional microarchitecture of the thymus plays a unique role in directing T cell lineage commitment and development. This is supported by the fact that, in contrast to fetal thymic organ cultures, thymic stromal cell monolayer cultures (TSMC) fail to support T lymphopoiesis. Nevertheless, OP9-DL1 cell monolayer cultures induce T lineage commitment and differentiation. Thus, the inability of TSMC to support T lymphopoiesis may be due to a loss of Notch ligand expression and/or function during culture. In this study, we report that, in contrast to fetal thymic organ cultures, TSMC fail to maintain expression of the Notch ligands, Delta-like (Dll) 1 and Dll4, and concomitantly lose the ability to support T lymphopoiesis. Importantly, ectopic re-expression of Dll1 or Dll4 is sufficient to restore the ability of TSMC to support T lymphopoiesis. These findings demonstrate that maintenance of endogenous Dll1 or Dll4 expression by thymic stromal cells is required for the commitment and differentiation of T cells in the absence of a three-dimensional microenvironment.     

Synergistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2 cytokine expression

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-gamma in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-gamma and IL-13. Analysis of the thymic subsets, CD4(-)CD8(-) (DN), CD4(+)CD8(+), CD4(+)CD8(-), and CD4(-)CD8(+) revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-gamma and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-gamma(+) cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 specifically induced CCR5 expression. The IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-gamma and IL-13 expression is dependent on Stat4 and NF-kappaB but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces significant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-gamma. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-gamma-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments.     

Total lymphoid irradiation leads to transient depletion of the mouse thymic medulla and persistent abnormalities among medullary stromal cells

Mice given multiple doses of sublethal irradiation to both the thymus and the peripheral lymphoid tissues showed major transient, and some persistent disruptions in general thymic architecture and in thymic stromal components. At 2 wk after total lymphoid irradiation (TLI), the thymus lacked identifiable medullary regions by immunohistochemical analyses. Medullary stromal cells expression MHC Ag or a medullary epithelial cell Ag, as well as medullary macrophages, were undetectable. Instead, the processes of cortical epithelial cells were observed throughout the entire thymus. Strikingly, thymocyte subsets with mature phenotypes (CD4+CD8- and CD4-CD8+) were present in the apparent absence of a medulla. This early, gross effect was rapidly reversed such that by 1 to 2 mo after TLI, medullary areas with MHC Ag-positive cells were evident. However, abnormalities in a subset of medullary stromal cells appeared to be more persistent. Medullary epithelial cells, identified by the MD1 mAb, were greatly reduced in number and abnormally organized for at least 4 mo after TLI. In addition, macrophages containing endogenous peroxidase activity, normally abundant in medullary regions, were undetectable at all times examined after TLI. Therefore, this irradiation regimen induced both transient and long term effects in the thymus, primarily in medullary regions. These results suggest that TLI may be used as an experimental tool for studying the impact of selective depletion of medullary stromal cells on the development of specific T cell functions.     

Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to sustained p53 activation

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1-/- and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1-/- DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal alphabeta T cell development.     

Expression of ets genes in mouse thymocyte subsets and T cells

The cellular ets genes (ets-1, ets-2, and erg) have been identified by their sequence similarity with the v-ets oncogene of the avian erythroblastosis virus, E26. Products of the ets-2 gene have been detected in a wide range of normal mouse tissues and their expression appears to be associated with cell proliferation in regenerating liver. In contrast, the ets-1 gene was previously shown to be more highly expressed in the mouse thymus than in other tissues. Because the thymic tissue contains various subsets of cells in different stages of proliferation and maturation, we have examined ets gene expression in fetal thymocytes from different stages of development, in isolated subsets of adult thymocytes, and in peripheral T lymphocytes. Expression of the ets-1 gene was first detected at day 18 in fetal thymocytes, corresponding to the first appearance of CD4+ (CD4+, CD8-) thymocytes, and reaches maximal/plateau levels of expression in the thymus at 1 to 2 days after birth. The ets-2 gene expression is detected at least 1 day earlier, coinciding with the presence of both double-positive (CD4+, CD8+) and double-negative (CD4-, CD8-) blast thymocytes and reaches maximal/plateau levels 1 day before birth. In the adult thymus, ets-1 and ets-2 mRNA expression is 10- to 8-fold higher respectively in the CD4+ subset than in the other subsets examined. Higher levels of p55 ets-1 protein were also shown to exist in the CD4+ subset. Because the CD4+ thymic subset is the pool from which the CD4+ peripheral, helper/inducer T cells are derived, the ets gene expression was examined in lymph node T cells. Both the CD4+ and the CD8+ T cells subsets had lower ets RNA levels than the CD4+ thymocytes. These results suggest that ets-2 and more particularly ets-1 gene products play an important role in T cell development and differentiation and are not simply associated with proliferating cells, which are observed at a higher frequency in fetal thymocytes, or dull Ly-1 (low CD5+), and double-negative (CD4-, CD8-) adult thymocytes. Selectively enhanced expression of ets-1 gene may be observed in thymic CD4+ thymocytes because these cells have uniquely encountered MHC class II or other Ag in the thymic environment. These cells may have been subsequently stimulated to activate the ets genes in conjunction with their differentiation of helper/inducer function(s) and expression of mature TCR.(ABSTRACT TRUNCATED AT 400 WORDS)