Somatic hypermutation and selection of B cells in thymic germinal centers responding to acetylcholine receptor in myasthenia gravis

The muscle weakness in myasthenia gravis (MG) is mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Production of these pathogenic autoantibodies is believed to be associated with germinal centers (GC) and anti-AChR-secreting plasma cells in the hyperplastic thymus of patients with early onset MG (EOMG). Here, we describe the repertoire of rearranged heavy chain V genes and their clonal origins in GC from a typical EOMG patient. Three hundred fifteen rearranged Ig V(H) genes were amplified, cloned, and sequenced from sections of four thymic GC containing AChR-specific B cells. We found that thymic GC contain a remarkably heterogeneous population of B cells. Both naive and circulating memory B cells undergo Ag-driven clonal proliferation, somatic hypermutation, and selection. Numerous B cell clones were present, with no individual clone dominating the response. Comparisons of B cell clonal sequences from different GC and known anti-AChR Abs from other patients showed convergent mutations in the complementarity determining regions. These results are consistent with AChR driving an ongoing GC response in the thymus of EOMG patients. This is the first detailed analysis of B cell clones in human GC responding to a defined protein Ag, and the response we observed may reflect the effects of chronic stimulation by autoantigen.     

Microenvironment of thymic myoid cells in myasthenia gravis

The microenvironment of myoid cells (MyCs) was studied in myasthenia gravis (MG) thymitis with lymphoid follicular hyperplasia (LFH) (nine cases) and with diffuse B cell infiltration (one case), and compared with findings in the thymuses of non-myasthenic control subjects (ten cases). Double immunostaining was used to demonstrate MyCs labelled by anti-desmin together with other thymic components such as keratin-positive epithelial cells, Ki-M 1-positive interdigitating reticulum cells (IDCs), Ki-M 4-positive follicular dendritic reticulum cells, Ki-M 6-positive macrophages, CD22-positive B-cells, CD1-positive cells, CD3-positive T-cells or HLA-DR-positive cells. Round or elongated MyCs were confined to the thymic medulla and were surrounded by CD3-positive T-cells and CD22-positive B-cells. In MG thymitis MyCs were localized in the vicinity of, but not inside germinal centres (GCs). MyCs were always HLA-DR-negative, but were invariably embedded in a cellular micromilieu with strong HLA-DR expression. A remarkable feature of MG thymitis was that the great majority of MyCs were in intimate contact with intramedullary IDCs. Morphometric studies confirmed that such contacts were significantly less frequent in thymuses from non-myasthenic subjects. This indicates that an IDC-dependent antigen-presenting process for T-cells may actively involve MyCs in MG thymitis.     

Expression of RAGE and HMGB1 in Thymic Epithelial Tumors,Thymic Hyperplasia and Regular Thymic Morphology

Recently, a role of the receptor for advanced glycation endproducts (RAGE) in myasthenia gravis was described. RAGE and its ligand high mobility group box 1 (HMGB1) play key roles in autoimmunity and cancer. To test whether these molecules are involved in patients with thymic abnormalities we applied immunohistochemical analysis in 33 cases of thymic epithelial tumors, comprising 27 thymomas and 6 thymic carcinomas, and 21 nonneoplastic thymuses. Both molecules were detected in neoplastic epithelial cells: RAGE staining was most intense in WHO type B2 thymomas and thymic carcinomas (p<0.001). HMGB1 nuclear staining was strongest in A and AB, and gradually less in B1 = B2>B3>thymic carcinoma (p<0.001). Conversely, HMGB1 cytoplasmic staining intensities were as follows: A and AB (none), B1 (strong), B2 (moderate), B3 and thymic carcinoma (weak); (p<0.001). Fetal thymic tissue showed a distinct expression of RAGE and HMGB1 in subcapsular cortical epithelial cells which was found in 50% of myasthenic patients. Furthermore RAGE and HMGB1 were expressed in thymocytes, macrophages, Hassall''s corpuscles, thymic medulla, and germinal center cells in myasthenic patients. Immunohistochemistry results were complemented by systemic measurements (immunosorbent assay): serum levels of soluble RAGE were significantly reduced in patients with epithelial tumors (p = 0.008); and in invasive tumors (p = 0.008). Whereas RAGE was equally reduced in thymic hyperplasia and epithelial tumors (p = 0.003), HMGB1 was only elevated in malignancies (p = 0.036). Results were most pronounced in thymic carcinomas. Thus, RAGE and HMGB1 are involved in the (patho-)physiology of thymus, as evidenced by differentiated thymic and systemic expression patterns that may act as diagnostic or therapeutic targets in autoimmune disease and cancer.     

Tumor necrosis factor-alpha and IFN-gamma expression in human thymus. Localization and overexpression in Down syndrome (trisomy 21).

In vitro studies suggest that TNF-alpha and IFN-gamma regulate thymocyte proliferation, but little evidence exists for the constitutive production of these cytokines in normal human thymus. In paired experiments, we examined frozen sections of postnatal human thymus from four control children and four age-matched children with Down syndrome (DS) (trisomy 21) for TNF-alpha and IFN-gamma mRNA expression using in situ hybridization. We studied thymuses from children with DS because this aneuploid condition is associated with a greatly increased incidence of infection and has abnormal thymic anatomy and patterns of thymocyte maturation. We found cells expressing constitutive levels of TNF-alpha mRNA in the trabeculae, corticomedullary junctions, and medulla of both control and DS thymuses and the number of these cells was an average of 3.9-fold higher in DS thymuses than in age-matched control thymuses. DS thymuses also contained an average of 3 fold higher numbers of cells with mast cell morphology, identified by toluidine blue histologic staining and electron microscopy. In both DS and control thymuses the mast cells colocalized with TNF-alpha mRNA-expressing cells. In addition, TNF-alpha protein- expressing cells, identified by immunohistochemistry, displayed a granular pattern of staining that is characteristic of mast cells. These results suggest that mast cells may be one source of TNF-alpha in human postnatal thymus. Discrete cells expressing IFN-gamma mRNA were distinctly localized to the cortical region of both DS and control thymuses and were 2.4-fold more abundant in DS thymuses than in the controls. Our results demonstrate, for the first time, the constitutive production and location of TNF-alpha and IFN-gamma in postnatal human thymus. The overexpression of both of these cytokines in DS thymuses suggests a dysregulation in cytokine production in DS and may provide an explanation for the abnormal thymic anatomy and thymocyte maturation associated with this syndrome.     

Effects of hypophyseal or thymic allograft on thymus development in partially decerebrated chicken embryos: expression of PCNA and CD3 markers

Changes in chicken embryo thymus after partial decerebration (including the hypophysis) and after hypophyseal or thymic allograft were investigated. Chicken embryos were partially decerebrated at 36–40 h of incubation and on day 12 received a hypophysis or a thymus allograft from 18-day-old donor embryos. The thymuses of normal, sham-operated and partially decerebrate embryos were collected on day 12 and 18. The thymuses of the grafted embryos were collected on day 18. The samples were examined with histological method and tested for the anti-PCNA and anti-CD3 immune-reactions. After partial decerebration, the thymic cortical and medullary compartments diminished markedly in size. Anti-PCNA and anti-CD3 revealed a reduced immunereaction, verified also by statistical analysis. In hypophyseal or grafted embryos, the thymic morphological compartments improved, the anti-PCNA and anti-CD3 immune-reactions recovered much better after the thymic graft, probably due to the thymic growth factors and also by an emigration of thymocytes from the same grafted thymus.Key words: hypophysectomy, hypophyseal and thymic allograft, chicken embryonal thymus, PCNA, CD3 markers.     

The human thymic microenvironment: cortical thymic epithelium is an antigenically distinct region of the thymic microenvironment

The thymic microenvironment is a complex tissue essential for normal T cell maturation. Prothymocytes in the subcapsular cortical (SCC) region of the thymus undergo cell division and migrate to the inner cortex. The majority of cortical thymocytes cease dividing and die, but a minority are exported to the periphery. We have previously shown thymic hormones in SCC and medullary thymic epithelium and have identified a monoclonal antibody (TE-4) that defines human endocrine thymic epithelium. However, no marker that selectively defines cortical thymic epithelium has been available. In this study, we have produced two monoclonal antibodies, TE-3A and TE-3B, raised against human thymic stroma that bind to an intracellular antigen in cortical but not medullary thymic epithelium. In double immunofluorescence assays in which we used anti-keratin, anti-thymosin alpha 1, and anti-endocrine thymic epithelium antibodies (TE-4, A2B5), TE-3+ SCC epithelium was TE-4+ and contained keratin and thymosin. alpha 1. In contrast, TE-3+ inner cortical epithelium was TE-4/A2B5 nonreactive and did not contain thymosin alpha 1. An ontogeny study of seven fetal and five neonatal thymuses demonstrated that expression of the TE-3 antigen was acquired at 10 wk fetal gestation. Using TE-3 antibody, we observed sequential stages of separation of cortical and medullary epithelium from 12 to 20 wk fetal gestation. In dysplastic (severe combined immunodeficiency disease) thymuses, strands of TE-3+ nonendocrine cells encircled nests of TE-4+ endocrine epithelium. Thus, human cortical thymic epithelium is antigenically distinct from endocrine medullary epithelium. Antibodies against the TE-3 antigen define an intracellular molecule that may reflect a specialized function of cortical thymic epithelium.     

Thymic epithelium is programmed to induce preleukemic changes in retrovirus expression and thymocyte differentiation in leukemia susceptible mice: studies on bone marrow and thymic chimeras

In the leukemia-prone AKR thymus, ecotropic and xenotropic-related viruses are expressed that generate leukemogenic recombinant viruses before the onset of leukemia. We have shown previously that (AKR X NZB)F1 hybrid mice do not develop leukemia because they severely restrict the expression of these retroviruses in their thymuses. The thymic microenvironment of the (AKR X NZB)F1 mice appeared to be of particular importance in determining this restriction, which was specified by an NZB-derived genetic influence. In the present study we analyze reciprocal thymus graft and irradiation bone marrow chimeras to establish that this influence is exerted by thymic reticuloepithelial cells. Prospective studies with thymic epithelial grafts from young mice show that the AKR thymic epithelium can simultaneously induce the amplified expression of retroviral genes, and changes in patterns of thymocyte differentiation that precede the development of leukemia, whereas the (AKR X NZB)F1 thymic epithelium is deficient in this regard.     

Insights into the Classification of Myasthenia Gravis

Background and Purpose

Myasthenia gravis (MG) is often categorized into thymoma-associated MG, early-onset MG with onset age <50 years, and late-onset MG with onset age ≥50 years. However, the boundary age of 50 years old between early- and late-onset MG remains controversial, and each category contains further subtypes. We attempted to classify MG from a statistical perspective.

Methods

We analyzed 640 consecutive MG patients using two-step cluster analysis with clinical variables and discrimination analysis, using onset age as a variable.

Results

Two-step cluster analyses categorized MG patients into the following five subtypes: ocular MG; MG with thymic hyperplasia (THMG); generalized anti-acetylcholine receptor antibody (AChR-Ab)-negative MG; thymoma-associated MG; and generalized AChR-Ab-positive (SP) MG without thymic abnormalities. Among these 5 subtypes, THMG showed a distribution of onset age skewed toward a younger age (p<0.01), whereas ocular MG and SPMG without thymic abnormalities showed onset age skewed toward an older age (p<0.001 and p<0.0001, respectively). The other 2 subtypes showed normal distributions. THMG appeared as the main component of early-onset MG, and ocular MG and SPMG without thymic abnormalities as the main components of late-onset MG. Discrimination analyses between THMG and ocular MG and/or SPMG without thymic abnormalities demonstrated a boundary age of 45 years old.

Conclusions

From a statistical perspective, the boundary age between early- and late-onset MG is about 45 years old.     

Migration of bone marrow cells toward the thymus in C57BL/Ka under fractionated irradiation

Fractionated whole body X-irradiation (4 X 1.75 Gy at weekly intervals) induces a high percentage of thymic lymphomas in C57BL/Ka mice. These tumors develop after a long latency period during which the thymic lymphopoiesis is deeply altered. In the present work, we test wether those modifications are due to lack of prothymocyte homing to preleukemic thymuses. Our results show that the preleukemic state of the thymus don't prevent the homing of normal marrow precursors grafted immediately after an irradiation of 4 Gy. Thus the alterations of thymic lymphopoiesis observed after a leukemogenic irradiation are not due to a modification in the thymus receptivity to thymocyte precursors.     

Age-related decrease of somatostatin receptor number in the normal human thymus

The thymus exhibits a pattern of aging oriented toward a physiological involution. The structural changes start with a steady decrease of thymocytes, whereas no major variations occur in the number of thymic epithelial cells (TEC). The data concerning the role of hormones and neuropeptides in thymic involution are equivocal. We recently demonstrated the presence of somatostatin (SS) and three different SS receptor (SSR) subtypes in the human thymus. TEC selectively expressed SSR subtype 1 (sst(1)) and sst(2A). In the present study we investigated whether SSR number is age related in the thymus. Binding of the sst(2)-preferring ligand (125)I-Tyr(3)-octreotide was evaluated in a large series of normal human thymuses of different age by SSR autoradiography and ligand binding on tissue homogenates. The score at autoradiography and the number of SSR at membrane homogenate binding (B(max)) were inversely correlated with the thymus age (r = -0.84, P < 0.001; r = -0.82, P < 0.001, respectively). The autoradiographic score was positively correlated with the B(max) values (r = 0.74, P < 0.001). Because the TEC number in the age range considered remains unchanged, the decrease of octreotide binding sites might be due to a reduction of sst(2A) receptor number on TEC. The age-related expression of a receptor involved mainly in controlling secretive processes is in line with the evidence that the major changes occurring in TEC with aging are related to their capabilities in producing thymic hormones. In conclusion, SS and SSR might play a role in the involution of the human thymus. These findings underline the links between the neuroendocrine and immune systems and support the concept that neuropeptides participate in development of cellular immunity in humans.     

Expression of preprotachykinin-A and neuropeptide-Y messenger RNA in the thymus.

The preprotachykinin-A gene, the common gene of mRNAs encoding both substance-P (SP) and neurokinin-A (NKA), was shown to be expressed in Sprague-Dawley rat thymus by detection of specific mRNA in gel-blot analyses. In situ hybridization revealed dispersed PPT-A-labeled cells in sections from rat thymus, with a concentration of grains over a subpopulation of cells in the thymic medulla. Also, neuropeptide-Y mRNA-expressing cells were found in the rat thymus, primarily in the thymic medulla. Rat thymic extracts contained SP-like immunoreactivity (SP-LI), and the major part of the immunoreactivity coeluted with authentic SP and SP sulfoxide standards. SP-LI was also detected in human thymus, which contained between 0.09-0.88 ng SP-LI/g wet wt. Evidence for translation of preprotachykinin-A mRNA in the rat thymus was obtained from the demonstration of NKA-LI in thymic cells with an epithelial-like cell morphology. Combined with previous observations on the immunoregulatory roles of tachykinin peptides and the existence of specific receptors on immunocompetent cells, the demonstration of intrathymic synthesis of NKA suggests a role for NKA-LI peptides in T-cell differentiation in the thymus.     

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.     

Phenotypic characterization of thymic prelymphoma cells of B10 mice treated with split-dose irradiation

Using an intrathymic injection assay on B10 Thy-1 congenic mice, it was demonstrated that thymic prelymphoma cells first developed within the thymuses from 4 to 8 days after split-dose irradiation and were detected in more than 63% of the test donor thymuses when examined at 21 and 31 days after irradiation. Moreover, some mice (25%) at 2 mo after split-dose irradiation had already developed thymic lymphomas in their thymuses. To characterize these thymic prelymphoma cells, the thymocytes from B10 Thy-1.1 mice 1 mo after irradiation were stained with anti-CD4 and anti-CD8 mAb and were sorted into four subpopulations. These fractionated cells were injected into the recipient thymuses to examine which subpopulation contained thymic prelymphoma cells. The results indicated that thymic prelymphoma cells existed mainly in CD4- CD8- and CD4- CD8+ thymocyte subpopulations and also in CD4+ CD8+ subpopulation. T cell lymphomas derived from CD4- CD8- prelymphoma cells had mainly CD4- CD8- or CD4- CD8+ phenotypes. T cell lymphomas developed from CD4- CD8+ prelymphoma cells mainly expressed CD4- CD8+ or CD4+ CD8+ phenotype. T cell lymphomas originating from CD4+ CD8+ prelymphoma cells were mainly CD4+ CD8+ but some CD4- CD8+ or CD4+ CD8- cells were also present. These thymic prelymphoma cells were further characterized phenotypically in relation to their expression of the marker defined by the mAb against J11d marker and TL-2 (thymus-leukemia) Ag, which is not expressed on normal thymocytes of B10.Thy-1.2 or B10.Thy-1.1 strain, but appears on the thymocytes of lymphomagenic irradiated mice. The results indicated that the prelymphoma cells existed in J11d+, TL-2+ cells.     

Expression of LIF in transgenic mice results in altered thymic epithelium and apparent interconversion of thymic and lymph node morphologies.

Leukemia inhibitory factor (LIF) is a cytokine involved in embryonic and hematopoietic development. To investigate the effects of LIF on the lymphoid system, we generated a line of transgenic mice that expresses diffusible LIF protein specifically in T cells. These mice display two categories of phenotype that were not previously attributed to LIF overexpression. First, they display B cell hyperplasia, polyclonal hypergammaglobulinemia and mesangial proliferative glomerulonephritis, defects similar to those described for transgenic mice overexpressing the functionally related cytokine, interleukin-6. Secondly, the LIF transgenic mice display novel thymic and lymph node abnormalities. In the thymus, cortical CD4+CD8+ lymphocytes are lost, while numerous B cell follicles develop. Peripheral lymph nodes contain a vastly expanded CD4+CD8+ lymphocyte population. Furthermore, the thymic epithelium is profoundly disorganized, suggesting that disruption of stroma-lymphocyte interactions is responsible for many observed defects. Transplantation of transgenic bone marrow into wild type recipients transfers both the thymic and lymph node defects. However, transplantation of wild type marrow into transgenic recipients rescues the lymph node abnormality, but not the thymic defect, indicating the thymic epithelium is irreversibly altered. Our observations are consistent with a role for LIF in maintaining a functional thymic epithelium that will support proper T cell maturation.     

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.     

Quantification of acetylcholinesterase-positive structures in human thymus during development and aging

Acetylcholinesterase (AChE) localization in the human thymus has been studied by biochemical and morphological methods during development and aging. The occurrence, the amount and the distribution of acetylcholinesterase and the changes with age were examined in 24 human thymuses. The whole human thymus was removed during autopsies in males of the following age-groups: prenatal of six months, new-born, infant, young, adult and elderly. The thymuses were weighed, measured and dissected: the microanatomical details were stained with Eosin-orange, nervous structures were identified by means of Bodian's method. Protein content was determined with biochemical methods. Histoenzymatical and biochemical demonstration of acetylcholinesterase was performed. The morphological results obtained were submitted to quantitative image analysis. Our results show that the thymic microenvironment changes with age; moreover, an increase of acetylcholinesterase-positive structures can be observed with age. Biochemical results are in agreement with morphological results and both are confirmed by the outcome of quantitative analysis of images. Acetylcholinesterase activity in human thymus may play a key role in thymic functions.     

Thymic medullary structures: Microscopical picture of the thymic medullary structures in children with congenital heart defects

The aim of this work is to describe the structure of the thymus, especially its medullary part, in children with congenital heart defects. It is known that development of the thymus and the heart is also influenced by neural crest cells. During the early development of the heart and the thymus cells proliferate and migrate to their primordia. It is known that inadequate cephalic neural crest contribution during development of pharyngeal pouch derivatives results in defective organogenesis of the face, the thymus, parathyroid glands and also the heart. We studied the structure of the thymus in children with congenital heart defects from 0 to 12 years of life at light microscopic and electron-microscopic levels. Thymuses of the patients were surgically removed in the Children’s Cardiocenter in Bratislava. The results of our study confirmed the differences in the medullary structures of thymuses with chosen diagnoses. Hassall’s corpuscles in the thymic medulla were various in size and also in structure and number. The special structures of the thymic medullary region in children with ventricular septal defects and defects of outflow of the heart were big cystic Hassall’s corpuscles. In comparison with a size of Hassall’s corpuscles in normal thymuses the size of Hassall’s corpuscles in studied thymuses suprisingly ranged between 100–250 μm.     

Implants of quail thymic epithelium generate permanent tolerance in embryonically constructed quail/chick chimeras

In situ implantation of a quail wing bud into a chick embryo at 4 days of incubation (E4) regularly results in the normal development of the implant followed by its acute rejection starting within two weeks post-hatching. If the epithelial thymic rudiments of the quail donor are implanted into the branchial arch area of the chick recipient after partial removal of its own thymic primordia, a chimeric thymus develops in the chick host and this induces tolerance to the quail wing by the chick recipient. The species identity of cells in chimeric thymuses was mapped using Feulgen-Rossenbeck' staining and immunolabelling with monoclonal antibodies directed against quail or chick B-L antigens. Certain lobes contained only chick cells both at the stromal and hemopoietic cell levels. Others had a quail epithelial stroma containing host hemopoietically derived cells. Only chimeras in which at least one third of the thymic lobes were chimeric showed permanent tolerance to the grafted wing. Since the two species exhibit distinct developmental rates, we decided to study the kinetics of thymic involution after birth. Although the changes in thymus weight and histological structure are fundamentally similar in quail and chick, those in the quail start about 7-8 weeks earlier. In the chimeric thymuses, the lobes whose epithelial cells were quail involuted at the rate of control quail showing no influence of the hemopoietic thymic compartment in this process. Tolerance induced by the thymic epithelium during embryogenesis and in early postnatal life was maintained after a profound involution of the quail thymic graft had occurred.