Thymic microenvironment and lymphoid responses to sublethal irradiation

Sublethal irradiation of the murine thymus has been a useful tool for depleting the thymus of dividing immature thymocyte subsets, to sequence thymocyte differentiation events occurring from radiation-resistant precursors. This massive reduction in thymocytes also represents a model in which the bidirectional interplay between the thymic stromal cells and lymphocytes can be investigated. The purpose of this study was thus twofold: to precisely map the initiation of thymopoiesis as a prelude to assessing the effects of injected mAb to novel thymic antigens; and to use a panel of mAbs to determine the alterations in the thymic stroma during the T-cell depletion and reconstitution phases. The striking finding from this study was that following T-cell depletion, there was a marked upregulation of specific stromal antigens, which retracted with the reappearance of T cells. Thus, following sublethal irradiation, there are modifications in the thymic microenvironment that may by necessary to support renewed thymopoiesis and the complete restoration of the thymus involved the synchronous development of both the stromal and lymphocytic components.     

Control of the thymic microenvironment by growth hormone/insulin-like growth factor-I-mediated circuits

The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo maturation, eventually leading to the migration of positively selected thymocytes to the T-dependent areas of peripheral lymphoid organs. This process occurs under the influence of the thymic microenvironment, by means of secretory polypeptides and cell-cell contacts. The thymic microenvironment is a tridimensional cellular network composed of epithelial cells (its major component), macrophages, dendritic cells, fibroblasts and extracellular matrix elements. The epithelial reticulum is a heterogeneous tissue, in which a particular lymphoepithelial structure has been isolated in vitro: the thymic nurse cell complex, which possibly creates particular microenvironmental conditions for thymocyte differentiation. Additionally, thymic nurse cells are useful tools to study mechanisms involved in intrathymic T cell migration, including neuroendocrine influences. Previous data showed that thymic hormonal function can be modulated by hormones and neuropeptides, including growth hormone. Interestingly, GH acts pleiotropically on the thymic epithelium increasing cell growth and expression of extracellular matrix ligands and receptors, the latter resulting in an enhancement of thymocyte adhesion to the epithelial cells and thymocyte release from thymic nurse cells. The role of GH on thymus development is further stressed by the findings obtained with GH-deficient dwarf mice. Besides the precocious decline in serum thymulin found in these animals, a progressive thymic hypoplasia occurs, with decreased numbers of CD4+CD8+thymocytes, both defects being largely restored by long-term GH treatment. The effects of GH in the thymus are apparently mediated by IGF-1. Enhancement of thymulin secretion induced by GH, as well as the stimulation of thymocyte adhesion to thymic epithelial cells can be prevented in vitro by treatment with antibodies for IGF-I or IGF-I receptor. Moreover, in both systems IGF-I alone can yield similar effects. Also, the enhanced concanavalin-A mitogenic response and IL-6 production by thymocytes observed in GH-treated mice can be detected in animals treated with IGF-I. Lastly, mouse substrains selected for high or low IGF-I circulating levels exhibited differential thymus developmental patterns correlating with IGF-I levels. A further conceptual aspect concerning the GH-IGF-I-mediated control of thymus physiology is the recent demonstration of an intrathymic production of these molecules, leading to the hypothesis that, in addition to the classical endocrine pathway, GH-IGF-I-mediated paracrine and autocrine pathways may also be implicated in the control of thymus physiology. In any case, such control is exerted pleiotropically, with modulation in the expression of several genes in different cell types of the organ. In this respect, it is exciting to imagine a role of GH-IGF-I loops in shaping the intrathymically generated T cell repertoire.     

Influence of water immersion stress on peripheral nerve recovery in the rat

Previously, we have shown that embryonic day 12 thymus anlage cultured alone cannot develop into the mature organ but degenerates. In the present study, we investigated the cause of this insufficient organogenesis of embryonic day 12 thymus anlage in organ culture. We cocultured embryonic day 12 thymus anlages with various cell lines as pellets formed by centrifugation. In coculture with fibroblastic cell lines, but not with thymic epithelial cell lines, embryonic day 12 thymus anlages developed to support full T cell differentiation, and expressed mature stromal cell markers, Ia and Kb. By pellet culture of thymus anlages and fibroblastic cell lines transfected with a beta-galactosidase expression vector, we analyzed the distribution of added fibroblastic cells in pellets. The added fibroblastic cells constituted neither thymic capsule nor septa but disappeared after about 2 weeks in culture. Moreover, immunohistochemical studies indicated that added fibroblastic cells were adjacent to mesenchymal cells of thymus anlage. Our results strongly suggest that added fibroblastic cells support the development of the thymus anlage through interaction with its mesenchymal cells.     

Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.     

Opportunistic fungal pneumonia

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive 35S-Na2SO4 and 3H-glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio 35S-sulfate/ H-glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin-like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions.     

Endometrial effects during hormone replacement therapy with a sequential oestradiol valerate/cyproterone acetate preparation

Different stages of thymus morphogenesis and thymocyte differentiation have been studied at the ultrastructural level in the lizard, Chalcides ocellatus. On stage 36 of embryonic development, the thymus primordium was composed principally of undifferentiated epithelial cells and some lymphoid stem-cells. From stage 37 to 38, the lymphoid stem-cells differentiate into lymphoblasts and then transform into typical lymphocytes. A clasmotosis phenomenon seems to be involved in this transformation. In the developing cortical regions, lymphoblasts accumulated rapidly, stretching the epithelial cells which become stellate in shape. From stage 39 to 40, a phase of intense proliferation occurs and numerous lymphocytes die in the thymic tissue and are phagocytosed by macrophages. On stage 41, the presence of interdigitating cells in the medullary area completed cortico-medullary differentiation. On neonatal and juvenile lizards, small cortical thymocytes differentiated and the thymus possessed all characteristic of an adult thymus. Thus, at birth, the histogenesis of the lizard thymus was achieved and the only further modification consisted in a gain of weight.     

Direct visualization of thymocyte apoptosis in neglect, acute and steady-state negative selection

During thymocyte differentiation, the majority of the developing cells die in situ by apoptosis and are subsequently removed by macrophages. DNA fragmentation is one of the hallmarks of apoptosis and can be detected in situ by TdT-mediated dUTP-biotin nick end labeling (TUNEL). We used TUNEL combined with immunohistology to determine the sites of thymocyte apoptosis in mice transgenic for a TCR (F5) which recognizes a peptide (NP68) of the influenza virus nucleoprotein (NP) presented on the MHC class I H-2Db molecule. Apoptosis due to neglect was studied in F5 mice expressing a neutral MHC haplotype (F5/H-2q) and in beta 2-microglobulin-deficient F5 mice (F5/ beta 2m+). In both cases, the frequency of apoptotic cells was similar to that seen in F5/H-2b mice and non-transgenic C57BI/10 mice. Antigen-induced apoptosis was studied in F5 mice after i.p. Injection of the cognate NP68 peptide and in F5/NP double-transgenic mice. Three hours after peptide injection, apoptosis was high throughout the thymus cortex and clusters of apoptotic cells formed due to tissue macrophage uptake, whereas the thymic medulla remained unaffected. Massive recruitment of inflammatory cells into the thymus was seen as early as 1 h after peptide injection. Nine hours after peptide injection changes were apparent in the cortical epithelium and, by 4 days, the cortical network had collapsed to give scattered, compacted epithelial cells. In contrast, in F5/NP double-transgenic mice, thymocyte apoptosis induced by cognate self-peptide was localized at the cortico-medullary junction with little change seen in the epithelium of the cortex.     

Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse

Infection with the human immunodeficiency virus (HIV) results in immunosuppression and depletion of circulating CD4+ T cells. Since the thymus is the primary organ in which T cells mature it is of interest to examine the effects of HIV infection in this tissue. HIV infection has been demonstrated in the thymuses of infected individuals and thymocytes have been previously demonstrated to be susceptible to HIV infection both in vivo, using the SCID-hu mouse, and in vitro. The present study sought to determine which subsets of thymocytes were infected in the SCID-hu mouse model and to evaluate HIV-related alterations in the thymic microenvironment. Using two different primary HIV isolates, infection was found in CD4+/CD8+ double positive thymocytes as well as in both the CD4+ and CD8+ single positive subsets of thymocytes. The kinetics of infection and resulting viral burden differed among the three thymocyte subsets and depended on which HIV isolate was used for infection. Thymic epithelial (TE) cells were also shown to endocytose virus and to often contain copious amounts of viral RNA in the cytoplasm by in situ hybridization, although productive infection of these cells could not be definitively shown. Furthermore, degenerating TE cells were observed even without detection of HIV in the degenerating cells. Two striking morphologic patterns of infection were seen, involving either predominantly thymocyte infection and depletion, or TE cell involvement with detectable cytoplasmic viral RNA and/or TE cell toxicity. Thus, a variety of cells in the human thymus is susceptible to HIV infection, and infection with HIV results in a marked disruption of the thymic microenvironment leading to depletion of thymocytes and degeneration of TE cells.     

A possible role for acetylcholine in the dialogue between thymocytes and thymic stroma

In this article we will review data suggesting that acetylcholine takes part in the mutual interplay between developing T cells and thymic epithelium, and thereby may influence the generation of the T-cell repertoire. In the first part we will recapitulate our findings according to which cholinergic agonists affect thymocyte apoptosis via a nicotinergic effect on thymic epithelial cells. In the second part we will present evidence that acetylcholine within the thymus is mainly derived from the thymocytes themselves, and that the production and release of this neurotransmitter is dependent on activation of thymic lymphocytes.     

The role of zinc in pre- and postnatal mammalian thymic immunohistogenesis

Histogenetically, the thymus is the primary lymphopoietic organ and provides an optimal microenvironment for the differentiation of T lymphocytes, independently of the influence of foreign antigens. Lymphocytes with diverse potential are produced in a protective microenvironment optimal for their maturation, whose dual cellular network is provided by endodermally derived RE cells and numerous ectomesenchymal cells derived from the neural crest. The full development of intrathymic hematopoiesis depends upon the successful completion of a series of well coordinated cellular interactions between widely divergent (in terms of origin) cells [epithelium (primitive pharynx); ectomesenchyrne (neural crest); and PHSCs (yolk sac, fetal liver)]. The cells of the thymic epithelial primordium do not proliferate in the absence of "inductive" interactions with the ectomesenchyme. Moreover, the nature of the mesenchyme determines the behavior of the thymic epithelial anlagen. The ectomesenchymal origin of chemotactic stem cell factor secretion, responsible for hemopoietic stem cell immigration, is a distinct possibility. The human thymus is a generalized hematopoietic tissue with between 7 to 9 weeks of ontogenesis. In human and dog fetuses various elements of mammalian hematopoiesis were identified intrathymically: B lymphocytes, plasma cells, erythropoietic and granulocytopoietic (neutrophils and eosinophils) cells, antigen presenting dendritic cells, and mast cells. Our light and ultrastructural (transmission and scanning), as well as immunocytochemical observations have established that during the embryonal and fetal period, the thymus is seeded by pluripotent, yolk sac derived PHSCs characterized by the following immunophenotype CD34+CD43+CD38-Lin-HLA-DR+CD69+. Stem cell c-kit tyrosine kinase (also referred to as mast cell growth factor, stem cell factor, or steel factor) in combination with autocrine and paracrine growth factors and cytokines (IL-3, IL-4, IL-5, IL-6, IL-7, G-CSF, etc.) stimulates myelopoiesis, including erythropoiesis, as well as lymphopoiesis. These hematopoietic growth factors are produced by activated lymphoblastic cells and stromal RE cells under the influence of immunoneuroendocrine regulation, supported by the finding that experimental or spontaneous, in vivo neural crest ablation during early mammalian ontogenesis always results in an abnormal development of the thymus, as well as the heart and great vessels, thyroid, and parathyroid glands.     

Naturally-occurring anti-thymocyte autoantibody which identifies a restricted CD4+CD8+CD3-/lo/int thymocyte subpopulation exhibits extensive polyspecificity

In this study, naturally-occurring, monoclonal IgM kappa anti-thymocyte autoantibodies from the neonatal inbred Balb/c mouse-derived hybridoma NMT-1 (NMT-1 mAb), previously reported to identify a restricted CD4+CD8+CD3/lo/int thymocyte subpopulation, have been shown to exhibit extensive polyspecificity. Using immunofluorescence, immunoblotting and antibody titration and competition ELISAs, NMT-1 mAbs exhibited polyspecific binding to 12 apparently structurally unrelated self and non-self antigens. The autoreactive component of the polyspecificity profile of NMT-1 mAbs encompassed reactivity to developmentally-related 14.5 and 18.3 kDa Thy-1 glycoforms expressed on a CD4+CD8+CD3-/lo/int thymocyte subpopulation. The autoreactivity profile of NMT-1 mAbs also included recognition of the heavy and light chains of mouse IgG1 and mouse cytokeratins within thymic medullary epithelium and basal epithelial cells of stratified squamous epithelium of mouse tongue, oesophagus, stomach, skin and vagina. Examination of the polyspecificity profile of NMT-1 mAbs was also undertaken using a panel of 23 antigens including heterologous proteins, phospholipids, haptens and bacterial antigens by antibody titration and competition ELISAs. Antibody titration ELISAs demonstrated that NMT-1 mAbs bound nine antigens including bovine carbonic anhydrase, ovalbumin, cardiolipin, phosphatidylserine, the haptens, DNP and FITC and the bacterial antigens including Escherichia coli beta-galactosidase and the toxoids from Corynebacterium tetani and Clostridium diphtheria. Competition ELISAs, based on the inhibition of NMT-1 mAb binding to antigens adsorbed to ELISA plate surfaces by inhibitor antigens in solution, demonstrated that NMT-1 mAb interactions were not dependent on multivalent binding. In these assays, NMT-1 mAbs recognized unmodified (native) epitopes on the solution phase forms of the protein antigens, including E. coli beta-galactosidase and toxoids from Corynebacterium tetani and Clostridium diphtheria, providing further evidence for the hypothesis that the binding of multiple, apparently unrelated, antigens by NMT-1 mAbs occurs via unique polyspecific antigen combining sites.     

Development of CD4 and CD8 single positive T cells in human thymus organ culture: IL-7 promotes human T cell production by supporting immature T cells

This paper describes novel model systems to study the development of human T cells. Fragments of neonatal human thymus (HUNT) can be cultured in vitro; the initial majority population of CD4, CD8 double-positive (DP) thymocytes is not maintained in organ culture. These cells are rapidly replaced by populations of CD4 or CD8 single-positive (SP) T cells. In addition, allogeneic thymic chimeras can be established by the addition of human cord blood (HUCB) mononuclear cells as a source of T progenitor cells to the organ cultures. Culture results in the acquisition of a mature SP T cell phenotype by the donor cells similar to that found when HUCB is allowed to develop in xenogeneic murine scid/scid fetal thymus organ culture. The number of immature and mature T cells produced by organ cultures can be differentially increased by the addition of exogenous IL-7, stem cell growth factor, IL-1, or GM-CSF. Anti-IL-7 antibody inhibits T cell production. Taken together, the results suggest that human T cell development occurs in these in vitro systems in a similar manner, regardless of the species origin of the thymic stromal cells in the culture, and that exogenous cytokines can be used to expand subpopulations of developing T cells.     

Influence of detergents on the function of cloned potassium channels

The human thymus is a lymphoepithelial organ in which T cells develop during fetal life. After maturation and selection in the fetal thymic microenvironment, T cells emigrate to peripheral lymphoid tissues such as the spleen, gut, and lymph nodes, and establish the peripheral T cell repertoire. Although the thymus has enormous regenerative capacity during fetal development, the regenerative capacity of the human postnatal thymus decreases over time. With the advent of intensive chemotherapy regimens for a variety of cancer syndromes, and the discovery that infection with the Human Immunodeficiency Virus (HIV) leads to severe loss of CD4+ T cells, has come the need to understand the role of the human thymus in reconstitution of the immune system in adults. During a recent study of the thymus in HIV infection, we observed many CD8+ T cells in AIDS thymuses that had markers consistent with those of mature effector cytotoxic T cells usually found in peripheral immune tissues, and noted these CD8+ effector T cells were predominately located in a thymic zone termed the thymic perivascular space. This article reviews our own work on the thymus in HIV-1 infection, and discusses the work of others that, taken together, suggest that the thymus contains peripheral immune cell components not only in the setting of HIV infection, but also in myasthenia gravis, as well as throughout normal life during the process of thymus involution. Thus, the human thymus can be thought of as a chimeric organ comprised of both central and peripheral lymphoid tissues. These observations have led us to postulate that the thymic epithelial atrophy and decrease in thymopoiesis that occurs in myasthenia gravis, HIV-1 infection, and thymic involution may in part derive from cytokines or other factors produced by peripheral immune cells within the thymic perivascular space.     

Thymic dendritic cells are primary targets for the oncogenic virus SL3-3

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70(+) cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70(+) cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70(+) cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70(+) dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.     

Antigenic stimuli do not influence thymic B lymphocytes: a morphological and functional study in germ-free and conventionally reared piglets

We have recently reported that thymic B lymphocytes (TBL) are the first B-cell subpopulation undergoing isotype switching to IgG and IgA during embryonic life. The aim of this study is to analyze the influence of antigenic stimulation on TBL location and activity using a germ-free (GF) newborn pig model, in which maternal antibodies and antigens do not affect B-cell development. Immunohistological analysis showed that TBL were disseminated mainly in the thymic medulla. There were no differences in the distribution of TBL, both in GF newborn piglets before and after colonization with Escherichia coli and in older conventionally reared (CONV) piglets. The number of immunoglobulin (Ig)-secreting cells measured by the ELISPOT method was not influenced by microflora and food antigens. IgM-positive cells secreting IgM and CD45RC-positive cells spontaneously producing IgM, IgG, and IgA were detected in newborn thymus. Our findings suggest that TBL differentiation and Ig switching to IgG and IgA-secreting cells is not influenced by external antigens and that the thymic microenvironment plays an important role in this process.     

Critical involvement of Tcf-1 in expansion of thymocytes

T cell maturation in Tcf-1(-/-) mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious blockades in the differentiation of CD4- 8- thymocytes are observed at two distinct stages (CD44+ 25+ and CD44- 25-), both of which are normally characterized by extensive proliferation. This lack of thymocyte expansion and/or differentiation was also observed when Tcf-1(-/-) progenitor cells from the aorta-gonad-mesonephros region (embryonic day 11.5), fetal liver (embryonic day 12.5/14.5), and fetal bone marrow (embryonic day 18.5) were allowed to differentiate in normal thymic lobes (fetal thymic organ cultures) or were injected intrathymically into normal recipients. Despite these apparent defects in thymocyte differentiation and expansion, adult Tcf-1(-/-) mice are immunocompetent, as they generate virus neutralizing Abs at normal titers. Furthermore, their peripheral T cells have an activated phenotype (increased CD44 and decreased CD62L expression) and proliferate normally in response to Ag or mitogen, suggesting that these cells may have arisen from the early wave of development during embryogenesis and are either long lived or have subsequently been maintained by peripheral expansion. As Tcf-1 is a critical component in the Wnt/beta-catenin signaling pathway, these data suggest that Wnt-like factors play a role in the expansion of double-negative thymocytes.     

Thymic overexpression of CD40 ligand disrupts normal thymic epithelial organization

We characterized the distribution of CD40 and CD40 ligand (CD40-L) in the adult and developing murine thymus. Before birth, CD40 was almost exclusively localized to scattered foci of medullary cells. By birth there was a dramatic upregulation of CD40 expression by cortical epithelial cells, which was accompanied by a consolidation of medullary epithelial foci. CD40-L+ thymocytes displayed a medullary location. Analysis of mice deficient in CD40-L expression indicated that CD40-L/CD40 interactions were not required for development of the medullary compartment. Overexpression of CD40-L targeted to thymocytes altered thymic architecture, as reflected by a dramatic loss of cortical epithelial cells, expansion of the medullary compartment, and extensive infiltration of the capsule with a mixture of CD3+ cells, B-cells, and macrophages/dendritic cells. Reconstitution of lethally irradiated normal mice with lck CD40-L bone marrow cells also resulted in loss of cortical epithelium and expansion of the medullary compartment. Disruption of the normal pattern of thymic architecture and epithelial differentiation as a consequence of increased intrathymic levels of CD40-L expression points to a role for CD40-L/CD40 interactions in the normal pattern of epithelial compartmentalization/differentiation within the thymic environment.