Tracing of cells of the avian thymus through embryonic life in interspecific chimeras

Differences in the structure of the interphase nucleus between two species of birds, the Japanese quail (Coturnix coturnix japonica) and the chick (Gallus gallus) has been used to distinguish cells from different origins in interspecies combinations. This biological cell marking technique was applied to thymus histogenesis. Using various combinations between components of quail and chick thymic rudiments, the respective contribution of endodermal epithelium, mesenchyme, and blood-borne extrinsic elements to the histogenesis of thymus was analyzed. It was demonstrated that the whole lymphoid population of the thymus is derived from immigrant blood-borne stem cells which are chemically attracted by the endoderm of the 3rd and 4th pharyngeal pouch. The latter is determined to differentiate into thymic epithelial reticulum as soon as the 15-somite stage, and is able to attract blood stem cells even when transplanted in an heterotopic position such as the ventral body wall of the embryo. It was shown that the thymic mesenchyme originates from the neural crest mesectoderm which colonizes early the 3rd and 4th branchial arches. It participates in the formation of perivascular mesenchyme, but does not give rise to lymphocytes. From heterospecific transplantations of quail thymuses into chick embryo (and inversely) at various stages of development is appeared that the thymic rudiment becomes attractive for lymphoid stem cells at a precise stage of its evolution for each species. The attractivity period lasts about 24 h for the quail and 36 h for the chick. Then, the inflow of stem cells becomes very low until the end of the incubation period. At this time, a second wave of lymphocytoblasts invades the thymus and the primitive embryonic lymphoid population is completely renewed around the hatching time. Competent thymic stem cells are present in the blood before and after the period of physiological thymic attractivity. The identity of basophilic cells appearing in the thymus during its histogenesis and lymphoid stem cells has been demonstrated from the analysis of quail-chick chimeric thymuses.     

Analysis of the first two waves of thymus homing stem cells and their T cell progeny in chick-quail chimeras

Chick-quail chimeras were used to study precursor/progeny relationships of hemopoietic stem cells (SC) that enter the embryonic thymus in waves to give rise sequentially to the TCR-1+, TCR-2+, and TCR-3+ lineages of T cells. The first wave of SC and their progeny were examined by grafting thymus from 9-d chick embryos (E9) into E3 quails. mAbs specific for chick T cell antigens were used to trace the development of T cells in the recipients. All three lineages of TCR-bearing cells were generated from the first wave of SC. The cortico-medullary transit time was several day shorter for the TCR-1 subpopulation than for the TCR-2 subpopulation, and the peripheral seeding of TCR-2 cells also occurred later in development. The duration of thymocyte production from the first wave of SC that entered the thymus was approximately 3 wk, during which gradual cortical to medullary replacement by second wave SC progeny occurred. When the latter was examined, after transplantation of E7 quail thymus into E3 chick embryos, a sequential generation pattern for the TCR-1 and TCR-2 cell progeny was not evident. Finally, recirculation of T cells to the thymus medulla was defined in this avian model.     

Growth of epithelial cells in the thymic medulla is under the control of mature T cells

Epithelial cells in the thymic medulla are conspicuous in normal adult mice, but sparse in the early fetal thymus and the thymus of adult T cell-deficient SCID mice. To examine whether growth of medullary epithelial cells (MEC) depends upon local contact with mature T cells, we used the finding that the SCID thymus is unusually permeable to mature T cells entering from the bloodstream. When SCID mice received multiple injections of mature lymph node T cells from birth, the thymus accumulated large numbers of mature TCR+ T cells of resting phenotype, but contained virtually no immature (CD4+8+) cells. The injected T cells localized in the medullary region of the thymus and led to marked regeneration of MEC. These and other data suggest that the growth of MEC is under the control of mature T cells. Placing MEC under T cell control might be a device for regulating the size and integrity of the medulla, especially during the phase of rapid thymic growth. Maintaining the cellular components of the medulla in proper balance could be critical for ensuring efficient self tolerance induction.     

STUDIES ON THYMUS FUNCTION : I. COOPERATIVE EFFECT OF THYMIC FUNCTION AND LYMPHOHEMOPOIETIC CELLS IN RESTORATION OF NEONATALLY THYMECTOMIZED MICE

Immunological restoration of 45-day old, neonatally thymectomized C3Hf mice by treatment with humoral thymic function (thymoma grafts, thymus or thymoma in diffusion chambers) ranges from 0 to 12% and is difficult to acheive. When small numbers (5–20 x 10⁶) of young adult lymphohemopoietic cells, ineffective by themselves, are given in association with humoral thymic function, a cooperative effect is observed and restoration ranges from 30 to 60%. With a particular cell dosage (20 x 10⁶), effectivity for cooperation with thymic function was the following in decreasing order: spleen, lymph nodes, thoracic duct cells, bone marrow, blood leukocytes, thymus, and Peyer's patch cells. Comparable results were obtained using spleen, thymus, and hemopoietic liver from newborn donors in association with thymic function. For similar cell dosages, newborn thymus cells were more effective than adult thymus in their cooperative effect with thymic function. Dispersed thymus cells in association with young adult bone marrow or newborn hemopoietic liver cells showed no synergism for the cooperative effect with thymic function in the present model. Using hemiallogeneic cells (F₁ hybrid into parent) it was possible to show that restoration was mediated by proliferative expansion of the injected cells. This was indicated by specific tolerance to tissues of the other parental strain and by cellular chimerism, especially of lymphoid tissues, as indicated by chromosome markers and absence of significant numbers of immunocompetent cells of host origin. A population of paritally differentiated cells of hemopoietic origin, termed postthymic, sensitive to humoral activity of the thymus and present in the lymphohemopoietic tissues of adult and newborn mice is postulated to explain our results. These cells are postthymic and thymus dependent in the sense that they already received thymic influence, probably through traffic, and are incapable of self-renewal in absence of the thymus. Sensitivity to humoral activity of the thymus is characterized by proliferative expansion and/or a differentiative process eventually leading to larger numbers of competent cells.     

STUDIES ON THYMUS FUNCTION : II. COOPERATIVE EFFECT OF NEWBORN AND EMBRYONIC HEMOPOIETIC LIVER CELLS WITH THYMUS FUNCTION

Significant immunological restoration of 45-day old, neonatally thymectomized C3Hf mice was obtained by the cooperation of syngeneic newborn or embryonic hemopoietic liver cells with thymic function. Thymic function or cells alone are almost ineffective or restore approximately 10% of the animals. Newborn liver cells are effective in association with thymus grafts or humoral thymic function (thymoma grafts and thymus or thymomas in diffusion chambers). Embryonic liver cells are ineffective, even in large numbers, when associated with humoral thymic function. On the other hand, embryonic liver cells are effective in the cooperative effect only in association with viable thymus grafts, preferably syngeneic, whether the grafts were placed subcutaneously, intraperitoneally, or under the kidney capsule. Dispersed viable thymic cells are ineffective in association with embryonic liver cells. Cells capable of cooperating with humoral thymic function start to appear to embryonic liver by day 19–21 of gestation and are detectable until day 5–6 postbirth. Embryonic hemopoietic liver cells from 12 to 18 days of gestation contain cells capable of cooperation only with viable free thymus grafts and not with humoral thymic function. A prethymic cell population of partially differentiated cells of hemopoietic origin, insensitive to humoral activity of the thymus but requiring thymic stroma and traffic through the thymus is postulated to explain our results. This population of prethymic cells can become postthymic through this process and eventually develop into competent cells. Postthymic cells are characterized by their sensitivity to humoral activity of the thymus and by their wide distribution in the lymphohemopoietic tissues of newborn and young adult mice.     

Characterization of human thymic lymphocytes forming rosettes with stromal cells.

The interaction of thymic lymphocytes and stromal cells is believed to be important for T cell development in thymus. In this study, thymic rosettes (TR), which are cell-cell complexes of thymic lymphocytes and stromal cells, were isolated from human thymic tissue, and were characterized. Treating human thymus with collagenase in mild condition, human TR were successfully isolated. Subsequently, TR were purified by the 1G sedimentation method. Human TR consisted of a stromal cell in center surrounded by lymphocytes. The stromal cells were positive for CD14, CD11b, and HLA-DR but negative for thymic epithelial cell specific mAb, UH-1, suggesting that they are macrophage/dendritic cells. The lymphocytes which formed TR (TRL) were mainly double positive (CD4+CD8+) and CD1+ cells, and few of them expressed bright CD3, indicating that TRL are in the intermediate maturation stage. TRL expressed activation markers (Ta1 and HLA-DR) in a significantly higher percentage of cells than did unselected thymocytes. Blocking test revealed that CD11a and CD2 are involved in the binding of TRL and the stromal cells as adhesion molecules.     

Monoclonal antibody against human T cell leukemia virus p19 defines a human thymic epithelial antigen acquired during ontogeny

Using monoclonal antibody 12/1-2 against a 19,000-dalton human T cell leukemia virus (HTLV) protein (anti-p19), previously demonstrated to be reactive with HTLV-infected human cells, but not in numerous other uninfected cells, we found a reactive antigen to be expressed on the neuroendocrine component of human thymic epithelial cells but not on any other normal epithelial or neuroendocrine human tissues. Moreover, this reactive antigen is acquired on neuroendocrine thymic epithelium during thymic ontogeny--first appearing on fetal thymic epithelial cells between 8 and 15 wk gestation. While only a portion of thymic epithelial cells in the subcapsular cortical region of 15- and 24-wk fetal thymuses contained anti-p19+ epithelial cells, the entire subcapsular cortical region of newborn thymus epithelium was anti-p19+. By age 3 yr, normal subjects' entire subcapsular cortical and medullary thymic epithelium was anti-p19+. Using antibody against HTLV core protein, p24, and c-DNA probes for HTLV DNA, neither HTLV-specific p24 protein nor proviral DNA could be demonstrated in anti-p19+ thymic epithelial tissue. However, thymic epithelial extracts, disrupted HTLV extracts, as well as purified HTLV p19 antigen all inhibited the binding of anti-p19 antibody to thymic epithelium. Thus, anti-p19 may recognize a determinant on an HTLV-encoded 19,000-dalton structural protein that is shared by human thymic epithelium. Alternatively, anti- p19 defines a host encoded protein that is selectively expressed by normal thymic epithelium, and is induced to be expressed in HTLV- infected malignant T cells.     

Identification of human and rodent thymic epithelium using tetanus toxin and monoclonal antibody A2B5.

Using a monoclonal antibody (A2B5), which binds to GQ ganglioside, and tetanus toxin, which binds to GD and GT gangliosides, distinct regions of human and rodent thymic epithelial cells have been identified. The lymphoid elements of the thymus do not bind A2B5 or tetanus toxin. The A2B5 and tetanus toxin-binding cells form a network of thymic epithelial cells throughout the thymic subcapsular cortex and thymic medulla and contain thymopoietin and thymosin alpha-1.     

Thymus in myasthenia gravis. Isolation of T-lymphocyte lines specific for the nicotinic acetylcholine receptor from thymuses of myasthenic patients.

The thymus is believed to play a central role in the pathogenesis of Myasthenia gravis (MG). According to a previous hypothesis, MG is initiated within the thymus by immunogenic presentation of locally produced nicotinic acetylcholine receptor (AChR) to potentially autoimmune T cells. Data of 10 consecutive MG patients demonstrate two critical features of MG thymuses that support the concept of intrathymic activation of autoreactive, AChR-specific lymphocytes. Morphologically, the thymuses showed lympho-follicular hyperplasia in nine cases and benign thymoma in one case. The paramount feature revealed by immunohistological double marker analyses was the intimate association of myoid cells (antigen producing) with interdigitating reticulum cells (potentially antigen presenting cells), both of which were surrounded by T3+ lymphocytes in thymus medulla. All 10 thymuses contained T lymphocytes reactive with AChR. This was in contrast to the peripheral immune compartment (blood) where in only 3 of 10 patients, significant T cell responses to AChR were observed. AChR-specific T cell lines could be established from 8 of 10 thymuses, all members of the helper/inducer subset as indicated by the expression of markers T3 and T4.     

Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development

Dendritic cells (DCs) in the thymus (tDCs) are predominantly accumulated in the medulla and contribute to the establishment of self-tolerance. However, how the medullary accumulation of tDCs is regulated and involved in self-tolerance is unclear. We show that the chemokine receptor XCR1 is expressed by tDCs, whereas medullary thymic epithelial cells (mTECs) express the ligand XCL1. XCL1-deficient mice are defective in the medullary accumulation of tDCs and the thymic generation of naturally occurring regulatory T cells (nT reg cells). Thymocytes from XCL1-deficient mice elicit dacryoadenitis in nude mice. mTEC expression of XCL1, tDC medullary accumulation, and nT reg cell generation are diminished in Aire-deficient mice. These results indicate that the XCL1-mediated medullary accumulation of tDCs contributes to nT reg cell development and is regulated by Aire.     

RANK signals from CD4(+)3(-) inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla

Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4(+)3(-) inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4(+)3(-) cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire(+) mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80(-)Aire(-) mTEC progenitors into CD80(+)Aire(+) mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire(+) mTECs and highlight a previously unrecognized role for CD4(+)3(-)RANKL(+) inducer cells in intrathymic self-tolerance.     

Early progression of thymocytes along the CD4/CD8 developmental pathway is regulated by a subset of thymic epithelial cells expressing transforming growth factor beta

Precursor cells differentiate into mature CD4+ and CD8+ T cells in the inductive environment of the thymus by undergoing a series of distinct developmental steps marked by expression of the coreceptor molecules CD4 and CD8. Among the earliest cells to enter the CD4/CD8 developmental pathway are CD4-CD8lo precursors cells that differentiate into CD4+CD8+ thymocytes. Here we show that differentiation of precursor cells into CD4+CD8+ thymocytes requires at least one cell division and that their progression through a cell cycle is specifically retarded in the thymus by interaction with thymic epithelial cells that express transforming growth factor beta (TGF- beta) proteins. We also demonstrate that TGF-beta proteins, either in solution or bound to cell membranes, can regulate cell cycle progression and differentiation of CD4-CD8lo precursor cells into CD4+CD8+ thymocytes. The regulatory effect of TGF-beta is specific for CD4-CD8lo precursor cells as TGF-beta proteins do not regulate the earlier generation of CD4-CD8lo precursor cells from CD4-CD8- thymocytes. Finally, we demonstrate that TGF-beta proteins are expressed in vivo in the intact thymus on subcapsular and cortical thymic epithelium where they can contact developing CD4-CD8lo precursor cells. Thus, thymic epithelial cells expressing TGF-beta proteins can actively regulate the rate at which CD4+CD8+ thymocytes are generated from CD4-CD8lo precursor cells.     

Differential requirement for mesenchyme in the proliferation and maturation of thymic epithelial progenitors

Formation of a mature thymic epithelial microenvironment is an essential prerequisite for the generation of a functionally competent T cell pool. It is likely that recently identified thymic epithelial precursors undergo phases of proliferation and differentiation to generate mature cortical and medullary thymic microenvironments. The mechanisms regulating development of immature thymic epithelial cells are unknown. Here we provide evidence that expansion of embryonic thymic epithelium is regulated by the continued presence of mesenchyme. In particular, mesenchymal cells are shown to mediate thymic epithelial cell proliferation through their provision of fibroblast growth factors 7 and 10. In contrast, differentiation of immature thymic epithelial cells, including acquisition of markers of mature cortical and medullary epithelium, occurs in the absence of ongoing mesenchymal support. Collectively, our data define a role for mesenchymal cells in thymus development, and indicate distinct mechanisms regulate proliferation and differentiation of immature thymic epithelial cells. In addition, our findings may aid in studies aimed at developing strategies to enhance thymus reconstitution and functioning in clinical certain contexts where thymic epithelial cell function is perturbed.     

Cathepsin L regulates CD4+ T cell selection independently of its effect on invariant chain: a role in the generation of positively selecting peptide ligands

CD4+ T cells are positively selected in the thymus on peptides presented in the context of major histocompatibility complex class II molecules expressed on cortical thymic epithelial cells. Molecules regulating this peptide presentation play a role in determining the outcome of positive selection. Cathepsin L mediates invariant chain processing in cortical thymic epithelial cells, and animals of the I-A(b) haplotype deficient in this enzyme exhibit impaired CD4+ T cell selection. To determine whether the selection defect is due solely to the block in invariant chain cleavage we analyzed cathepsin L-deficient mice expressing the I-A(q) haplotype which has little dependence upon invariant chain processing for peptide presentation. Our data indicate the cathepsin L defect in CD4+ T cell selection is haplotype independent, and thus imply it is independent of invariant chain degradation. This was confirmed by analysis of I-A(b) mice deficient in both cathepsin L and invariant chain. We show that the defect in positive selection in the cathepsin L-/- thymus is specific for CD4+ T cells that can be selected in a wild-type and provide evidence that the repertoire of T cells selected differs from that in wild-type mice, suggesting cortical thymic epithelial cells in cathepsin L knockout mice express an altered peptide repertoire. Thus, we propose a novel role for cathepsin L in regulating positive selection by generating the major histocompatibility complex class II bound peptide ligands presented by cortical thymic epithelial cells.     

Bone marrow origin of Ia-positive cells in the medulla rat thymus

Irradiated rats were reconstituted with bone marrow from F1 hybrids. Ia antigen of donor-bone marrow origin was detected by an immunoperoxidase technique on cryostat sections and found predominantly in the medulla of rat thymus 2 wk after reconstitution. These Ia-bearing cells increased in number with time after reconstitution, but the Ia on the cortical epithelial cells remained of host origin. The nature of the bone marrow-derived cells and their implication for major histocompatibility complex restriction are discussed.     

Clonal deletion induced by either radioresistant thymic host cells or lymphohemopoietic donor cells at different stages of class I-restricted T cell ontogeny.

Major histocompatibility complex (MHC) products and self-antigens expressed in the thymus determine the repertoire of mature alpha/beta T cells. While positive selection of self-MHC-restricted T cells is directed by MHC molecules expressed by thymic epithelial cells, negative selection depends to a large extent on self-antigens presented by lymphohemopoietic cells. However, radioresistant components of the thymus also influence negative selection, but it remains controversial whether this is accomplished by clonal deletion, clonal anergy, or other mechanisms. In this study, T cell development in mice expressing a transgenic T cell receptor (TCR) specific for lymphocytic choriomeningitis virus (LCMV) plus H-2Db was analyzed in the presence or absence of the viral antigen. A novel approach to analyze the thymic tissue requirements for negative selection was possible by comparing thymocyte selection in H-2Db versus H-2Dbm13 mice, since the latter allowed positive selection but not LCMV-specific deletion of transgenic TCR-expressing thymocytes. In irradiation bone marrow chimeras expressing the restriction element for negative selection (H-2Db) on host tissue, we show that radioresistant recipient cells in the thymus deleted developing T cells at an early stage of differentiation. In contrast, chimeras expressing H-2Db on lymphohemopoietic donor cells showed clonal deletion at a later stage during ontogeny.     

Thymoma epithelial cells secrete thymic hormone but do not express class II antigens of the major histocompatibility complex

17 thymomas were studied by indirect immunofluorescence for the presence of thymic hormones and antigens of the major histocompatibility complex (MHC). The thymoma epithelial cells (specifically identified by their keratin content) contained thymic hormones (thymulin and thymosin alpha 1), a finding corroborated by the observation of elevated thymulin serum levels. In contrast with normal or hyperplastic thymuses, thymoma epithelial cells did not express HLA-DR and HLA-DC antigens as assessed by immunofluorescence as well as immunoblot analyses. Conversely, MHC class I antigens (HLA-ABC) were normally expressed. Thus, we conclude that thymoma epithelial cells are endocrinologically active but are defective for the expression of some MHC products (class II molecules) known to play an essential role in intrathymic T cell differentiation.     

Ia expression by vascular endothelium is inducible by activated T cells and by human gamma interferon

We have used monoclonal antibody binding, measured by radioimmunoassay, fluorescence flow cytometry, and ultrastructural immunocytochemistry, to measure expression of Ia antigens on cultured human umbilical vein endothelial (HUVE) cells. Under standard culture conditions, HUVE cells do not express Ia antigens. However, treatment of primary HUVE cultures with phytohemagglutinin induces the expression of Ia antigens. Every endothelial cell in the culture becomes Ia-positive and endothelial cells appear to synthesize Ia. HLA-A,B is concomitantly increased. The expression of Ia appears to be mediated by T cells because (a) pretreatment of primary HUVE cultures with OKT3 plus complement blocks the action of the lectins but not of medium conditioned by lectin- activated peripheral blood mononuclear cells; (b) co-culture of endothelial cells with allogeneic T cells, in the absence of lectin, also induces endothelial Ia; and (c) human immune (gamma) interferon, produced by Chinese hamster ovary cells transfected with the human gamma interferon gene, directly induces endothelial Ia. During co- culture with lymphocytes, about one-third of the endothelial cells are Ia-positive after 24 h and all of the endothelial cells are Ia-positive by 72 h. Proliferation of allogeneic T cells starts by 96 h and peaks at 144 h. Thus, endothelial Ia appears sufficiently early to be a determinant for the proliferation of allogeneic T cells. Inducible expression of Ia by endothelium may be important both for allograft rejection and for recruitment of circulating T cells into the site of an immune response.     

Thymic B lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, alpha actinin, or actin

Striational autoantibodies (StrAb), which react with elements of skeletal muscle cross-striations, occur frequently in patients with thymoma associated with myasthenia gravis (MG). Dissociated thymic lymphocytes from 22 of 72 MG patients secreted StrAb when cultured with PWM. A high yield of EBV-transformed B cell lines was established from thymus, thymoma, and peripheral blood of seven patients with MG, but clones secreting StrAb arose only from the three patients who had StrAb in their sera. The monoclonal StrAb bound to A bands or I bands in skeletal muscle of human, rat, and frog. One bound to mitochondria in addition to myofibrillar I bands. None bound to nuclei, smooth muscle, or gastric mucosal cells. In immunoblot analyses and ELISAs the monoclonal StrAb bound to muscle and nonmuscle isotypes of myosin, alpha actinin, and/or actin. All bound to contractile proteins common to thymus and muscle, and one selectively immunostained epithelial cells of the thymic medulla. From these antigenic specificities we suggest that StrAb might arise as an immune response directed against the cytoskeletal anchoring proteins associated with nicotinic acetylcholine receptors in thymic epithelial cells undergoing neoplastic transformation to thymoma.     

Thymic origin of embryonic intestinal gamma/delta T cells

Current evidence suggests both thymic and extrathymic origins for T cells. Studies in mice favor an in situ origin for a prominent population of intestinal intraepithelial lymphocytes that express gamma/delta T cell receptor (TCR). This developmental issue is explored in an avian model in which the gamma/delta lymphocytes constitute a major T cell subpopulation that is accessible for study during the earliest stages of lymphocyte development. In the chick embryo, cells bearing the gamma/delta TCR appear first in the thymus where they reach peak levels on days 14-15 of embryogenesis, just 2 d before gamma/delta T cells appear in the intestine. Using two congenic chick strains, one of which expresses the ov antigen, we studied the origin and kinetics of intestinal colonization by gamma/delta T cells. The embryonic gamma/delta+ thymocytes homed to the intestine where they survived for months, whereas an embryonic gamma/delta- thymocyte population enriched in thymocyte precursors failed to give rise to intestinal gamma/delta+ T cells. Embryonic hemopoietic tissues, bone marrow, and spleen, were also ineffective sources for intestinal gamma/delta+ T cells. Intestinal colonization by gamma/delta+ thymocytes occurred in two discrete waves in embryos and newly hatched birds. The data indicate that intestinal gamma/delta T cells in the chicken are primarily thymic migrants that are relatively long-lived.