Feedback regulation of the secretion of a thymic hormone (thymulin) by human thymic epithelial cells in culture.

The production of the thymic hormone, thymulin (FTS), was studied in primary cultures of human thymic epithelium by immunofluorescence using monoclonal anti-thymulin antibodies. The number of thymulin-containing cells and the thymulin level in the culture supernatant increased gradually during the culture. Addition of synthetic thymulin to the culture medium reduced significantly the increase of thymulin-containing cells. Conversely, addition of monoclonal anti-thymulin antibody from the beginning of the culture exacerbated the spontaneous increase of thymulin-containing cells and abrogated the effects of thymulin. Combined with similar data previously reported in vivo, these results demonstrate that thymulin is actively produced by cultured thymic epithelial cells and that its synthesis can be down-regulated by the hormone itself.

A zinc-dependent epitope on the molecule of thymulin, a thymic hormone.

Thymulin is a nonapeptide hormone produced by thymic epithelial cells. Its biological activity is strictly dependent on the presence of the metal zinc in the molecule. Antithymulin monoclonal antibodies have been produced against either the synthetic (AS1) or the natural intraepithelial (AE1) molecule. These monoclonal antibodies were screened for their abilities to inhibit the zinc-dependent biological activity of the hormone and were shown to bind to thymic epithelial cells. By using biological and immunofluorescence assays, the two antibodies were shown to recognize exclusively the zinc-coupled thymulin molecule. Other antithymulin antibodies screened by RIA or ELISA (using a zinc-deprived substrate) recognized a zinc-independent epitope on the thymulin molecule. These data indicate the existence of a zinc-specific conformation on the thymulin molecule. They are in agreement with NMR studies showing that the zinc-containing hormone has a unique structure.

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.

Interferon-gamma modulates HLA class II antigen expression on cultured human thymic epithelial cells.

Cultures of human thymic epithelial cells (TEC) were tested for the expression of HLA class I (A, B, C) and class II (DR and DC) antigens by indirect immunofluorescence. The epithelial nature of the cells was proven by using an antikeratin antiserum. A high level of expression (close to 100% positive cells) of HLA class I antigens was observed on TEC at the beginning of the culture and remained unchanged for up to 12 days. In contrast, HLA class II antigen expression (85% DR+ and 75% DC+ cells on day 2) decreased gradually and reached very low levels (less than 5% DR+ or DC+) by day 7 of culture. This loss of class II antigen expression was not seen when cultures were performed in the presence of supernatants from activated T cells containing interferon-gamma (IFN-gamma). Furthermore, the presence of recombinant IFN-gamma (rIFN-gamma) in the medium from the onset of culture maintained HLA-DR and DC antigen expression on a high number of cells (comparable to that observed on day 2 of culture). A large percentage of rIFN-gamma-treated cells also showed intracytoplasmic HLA-DR antigen expression. Addition of rIFN-gamma at various times after the onset of the culture led to a reinduction of DR and DC antigen expression. This effect of rIFN-gamma was observed in 48 hr with concentrations as low as 10 IU/ml and was apparently specific for this IFN species, in that rIFN-alpha was unable to modify HLA class II antigen expression at concentrations up to 1000 IU/ml. The increased expression of HLA class II antigen was truly due to induction in individual TEC, rather than selection of class II-positive cells, because induction under the influence of IFN-gamma was reversible and occurred in the absence of proliferation in mitomycin-treated or gamma-irradiated cultures. Our results indicate that synthesis and membrane expression of class II HLA antigens are enhanced by IFN-gamma in TEC cultures. This finding raises the possibility that IFN-gamma participates in the mechanisms that assure the permanent expression of DR and DC antigens observed in TEC in vivo, with potentially important functional consequences in terms of education for self recognition.

Extracellular matrix of the human thymus: immunofluorescence studies on frozen sections and cultured epithelial cells.

The immunohistochemical detection of elements of the human thymic extracellular matrix in situ and in vitro is described. In the normal thymus, the intracapsular and intraseptal fibers were strongly labeled by anti-type I collagen antiserum. Basement membranes bordering the capsule, septae, and perivascular spaces were intensely stained by anti-type IV collagen, anti-fibronectin, and anti-laminin sera. In hyperplastic myasthenia gravis thymuses, the major changes consisted of discontinuities of the basement membrane adjacent to clusters of epithelial (keratin-containing) cells, among which an unusual connective framework (densely labeled by all the antisera) was observed. In vitro, most epithelial cells were strongly labeled by antifibronectin serum and to a lesser extent by the anti-type IV collagen and anti-laminin sera. In addition, fibronectin, laminin, and type IV collagen were detected in the intercellular spaces bordering the epithelial cells in culture. Results show that thymic epithelial cells participate in the synthesis of extracellular matrix elements, which as a result of their localization and influence on epithelial cell growth, should be regarded as constitutive components of the thymic microenvironment.

[HLA and myasthenia. Subdivision in 3 categories]. / HLA et myasthéne. Subdivision en trois catégories.

Genetic susceptibility (HLA types), clinical and pathological findings, amount of acetylcholine receptor antibodies and T lymphocyte subpopulations were studied in 63 patients with Myasthenia Gravis (MG). The frequency of HLA-DR5 was increased among patients (0.50 versus 0.23 in controls, pc less than 0.01, relative risk 3.3) and that of HLA-DR3 previously described as associated with MG was slightly increased (0.31 versus 0.20 in controls). The relative frequencies of two T cell subpopulations (T4 helper and T8 suppressor/cytotoxic lymphocytes) were normal in HLA-DR5 positive patients while the ratio T4/T8 was increased in other MG patients, who were HLA-DR3 (p less than 0.005). The high rate was due to an increase in the absolute number of T4 lymphocytes (p less than 0.001). HLA-DR3 patients were mostly women with early onset of a severe form of the disease, marked by the presence of thymic follicular lymphoid hyperplasia. A third genetic susceptibility to this disease was recently described in patients treated with D-penicillamine, the antigenic frequency of HLA-Bw35, DR1 is significantly increased. These 3 types of association between HLA and myasthenia gravis can be related to three different physiopathological mechanisms: the first two are probably linked to individual immunity (inductor/suppressor disequilibrium), in the third association, the mechanism is immunopharmacological.

Thymic hormone-containing cells. V. Immunohistological detection of metallothionein within the cells bearing thymulin (a zinc-containing hormone) in human and mouse thymuses.

Using an immunofluorescence (IF) assay, the presence of metallothionein (MT) was investigated in sections of normal and pathologic human thymuses as well as in cultures of thymic epithelial cells. This protein, known to have a high binding affinity for class II B transitional metals, such as zinc, was detected in the epithelial component of the thymus. Moreover, double labeling experiments with the anti-MT and an anti-thymulin monoclonal antibody showed that all cells containing thymulin, a thymic hormone whose active structure is known to contain zinc, also exhibited large amounts of metallothionein. These results, together with the fact that zinc and thymulin have been detected in the same type of cell organelles, lead to the conclusion that the MT present in thymic epithelial cells might be involved in the mechanism of zinc storage in these cells, thus favoring the secretion of thymulin in its biologically active, zinc-containing form.

Thymic epithelial antigen, acquired during ontogeny and defined by the anti-p19 monoclonal antibody, is lost in thymomas.

The protein defined by the anti-p19 monoclonal antibody (raised against a 19-kilodalton protein from human T leukemia virus) and normally acquired during the ontogenesis of the human thymic epithelium was studied by indirect immunofluorescence in thymomas and thymic hyperplasias. Hyperplastic (and involuted) thymuses from patients with myasthenia gravis exhibited a clear-cut expression of this differentiation antigen exclusively in epithelial cells, thus qualitatively similar to what was observed in the normal thymus. In thymoma epithelial cells, however, this expression was strongly altered: in the 12 malignant thymomas studied, no anti-p19 reactivity was found. In the four benign cases, a variable number of epithelial cells expressed this antigen. These results suggest that the differentiation antigen of the human thymic epithelium defined by the anti-p19 monoclonal antibody is lost during the malignant transformation occurring in thymomas. Furthermore, this monoclonal antibody provides an additional tool for the study of the degree of dedifferentiation of thymomas.

Production of anti-thymulin (FTS) monoclonal antibodies by immunization against human thymic epithelial cells.

A monoclonal antibody specific for thymulin (FTS), a thymic hormone initially isolated from serum, was obtained by cell fusion using spleen cells from BALB/c mice immunized with cultured human thymic epithelial cells. Hybridomas were selected according to their capacity to produce antibodies binding specifically to thymic epithelial cells in culture (as assessed by indirect immunofluorescence) and their ability to absorb in vitro the biological activity of synthetic and natural hormone preparations and to induce in vivo the disappearance of endogenous circulating thymulin. In this way monoclonal antibodies were obtained that recognized a subpopulation of nonlymphoid cells on frozen sections of mouse and human thymuses. The epithelial nature of these cells was assessed using an antikeratin antiserum. The binding of the antibodies to thymic cells was completely abolished by its absorption with the synthetic hormone or normal (but not of thymectomized) mouse serum. The thymic specificity of the antibody was further confirmed by the complete absence of binding to sections of all the various lymphoid and epithelial organs examined (from both humans and mice). Double labeling experiments using the monoclonal antibody described above and a monoclonal antibody prepared by immunization with the synthetic peptide showed that the two antibodies bound to the same cell. These results provide further evidence for the exclusive presence of the thymic hormone thymulin in thymic epithelial cells.

The in vivo effects of corticosteroids on thymocyte subsets in myasthenia gravis.

The in vivo effects of corticotherapy on thymocyte subpopulations have been evaluated in patients with myasthenia gravis (MG). Ten patients receiving high-dose, long-term treatment were studied and compared with two control groups (MG untreated patients and normal age-matched subjects). In the treated group, the thymus was generally involuted; the percentage of OKT6+ or OKT4+T8+ thymocytes was profoundly decreased compared to controls. A significant percentage of OKT10 - cells was detected particularly among older patients, suggesting steroid-induced immigration. Conversely the percentage of more mature OKT3+ cells was increased. The balance between OKT4+T8- and OKT4-T8+ cells was unchanged in young patients (less than 40 years old) and increased in the older group. These data show that, as in the mouse, corticosteroids profoundly alter human thymocyte subsets.

Anti-AChR antibodies, thymic histology, and T cell subsets in myasthenia gravis.

The relationship between the titers of antibody against acetylcholine receptor (AChR) and T helper/suppressor balance (assessed by the OKT4/OKT8 ratio) were investigated in 74 patients with myasthenia gravis (MG). All patients with elevated AChR antibody titers (greater than 100 nM) had hyperplastic thymuses, while most patients with low or negative antibody titers (less than 1 nM) had involuted thymuses. All patients with thymoma had positive, though not very high, antibody titers. No correlation was found between anti-AChR antibody levels and OKT4/OKT8 ratios except for patients with thymoma. Thus, it appears that AChR antibody titers are more closely related to thymic pathology than to peripheral T cell imbalance. These results are consistent with the hypothesis giving a central role to thymic lymphocytes in the AChR antibody production, either as antibody producer B cells or helper T cells.

Depletion of helper/inducer T cells after thymectomy in myasthenic patients.

Using monoclonal anti-T-cell antibodies, we have studied peripheral blood T-cell subsets in 53 patients with myasthenia gravis before and after thymectomy (Tx). Before Tx, the mean OKT4/OKT8 ratio was higher in patients than in controls. Furthermore patients showed a high number of cells reacting simultaneously with the OKT4 and OKT8 antibodies. Shortly after surgery, the helper/suppressor ratio was increased in most of the patients, and the doubly reactive subset decreased to normal levels. However, 6 to 12 months after Tx the OKT4/OKT8 ratio was significantly decreased, particularly in patients showing clinical improvement. The percentage of total T cells was slightly but significantly reduced. A group of 14 patients studied more than 2 years after Tx presented very low OKT4/OKT8 ratios. Thymectomy in MG appears to lead to a gradual decrease of the T-helper subset which could contribute to its favorable effect on the course of the disease.

[In vitro regulation of the secretion of thymulin (STF) by human thymic epithelial cells]. / Régulation in vitro de la sécrétion de thymuline (FTS) par les cellules épithéliales thymiques humaines.

The presence of thymulin (formerly called Facteur Thymique Sérique, FTS) in cultured human thymic epithelial cells was studied using a monoclonal anti-thymulin antibody in an immunofluorescence technique. The percentage of thymulin-containing cells increases gradually as a function of time, from 10% at day 4 to 90% at day 14. Furthermore, this increase can be prevented by the addition of thymulin in the culture medium. These data suggest a feedback control of thymulin secretion by thymic epithelial cells.

The effects of immunomodulation on peripheral T cell subsets.

Monoclonal antibody assays were employed to monitor modifications induced in human peripheral lymphocyte subsets by thymectomy or the administration of a series of immunomodulating drugs: synthetic thymic factor, cimetidine or various combinations of anti-thymocyte globulin, azathioprine and steroids. In patients with myasthenia gravis, thymectomy produced a gradual progressive decrease in the elevated OKT4/OKT8 ratios associated with this disease until normal ratios were achieved after one year. Administration of synthetic thymic factor to three immunodeficient children for one month produced increased serum IgA levels accompanied by a normalization of proportions of total T cells and T cell subsets. Four of five uremic patients receiving cimetidine exhibited a marked increase in the percentage of OKT8+ T cells observed in subsequent blood samples with a concomitant increase in immature (OKT4+, OKT8+) lymphocytes that suggested an increase in release of such lymphocytes from the thymus. Assessment of 29 longterm renal allograft recipients by repeated T cell monitoring over an extended period of time confirmed the findings of other investigators that an increase in the OKT4+/OKT8+ ratio was predictive of subsequent allograft rejection episodes while subnormal OKT4+/OKT8+ ratios were indicative of possible cytomegalovirus or herpes virus infections.

[Assay of acetylcholine receptor antibodies in myasthenia gravis. A study of 329 sera (author's transl)]. / Le dosage des anticorps anti-récepteurs de l'acétylcholine dans la myasthénie. Etude de 329 sérums.

Antibodies directed against acetylcholine receptors were investigated in 329 sera by a radioimmunological technique using human muscle. Eighty-six per cent of patients with generalized myasthenia gave positive results. The percentage of positivity reached 90% when a receptor lipid extract was used. Fifty-six per cent of 25 sera from patients with ocular myasthenia were positive. Conversely, no antibody was detected in controls (other neurological diseases, normal subjects), except for two lupus patients. In addition to its diagnostic value, the assay of antibodies to acetylcholine receptors proved useful in the follow-up of myasthenic patients (notably neonates) treated by thymectomy, immunodepressants and plasmapheresis.

Evaluation of T cell subsets in myasthenia gravis using anti-T cell monoclonal antibodies.

Functional T cell subsets have been evaluated in the peripheral blood of patients with myasthenia gravis using monoclonal anti-T cell antibodies and a suppressor cell assay based on the suppression of the mixed-lymphocyte reaction by concanavalin A-activated lymphocytes. A significant decline of suppressor cells was found in a large proportion of patients, both by direct count using the anti-suppressor-cytotoxic T cell antibody (OKT8) and by the suppressor assay. Patients also showed an increase in immature T cells defined by their simultaneous reaction with the anti-helper cell (OKT4) and anti-suppressor cell (OKT8) antibody. Thymectomy tended to enhance the deficit in suppressor cells, and to induce the disappearance of double-labelled cells.

Thymic involution in pregnant mice. I. Characterization of the remaining thymocyte subpopulations.

Pregnancy-induced thymic atrophy was studied in mice during the course of syngeneic gestation and the post-partum period. Cortical thymocytes were greatly reduced in number as shown by the binding of fluorescein-labelled PNA. The pool of steroid-resistant (SR) medullary thymocytes appeared unchanged in pregnant mice when studied by means of a specific heteroantiserum (SRCA). Therefore, in pregnant mice, these two surface markers demonstrated that thymic atrophy was linked to steroid-sensitive (SS) cortical cell reduction. The presumed hydrocortisone resistance of the mother's remaining thymocytes is not related to a difference in the number of steroid receptors as determined by 3H-dexamethasone binding.