Cultures Of Thymic Cells Research Articles

Fibronectin-Functionalized Fibrous Meshes as a Substrate to Support Cultures of Thymic Epithelial Cells.

Thymic epithelial cells (TECs) are the main regulators of T lymphocyte development and selection, requiring a three-dimensional (3D) environment to properly perform these biological functions. The aim of this work was to develop a 3D culture substrate that allows the survival and proliferation of TECs. Thus, electrospun fibrous meshes (eFMs) were functionalized with fibronectin, one of the major extracellular matrix (ECM) proteins of the thymus. For that, highly porous eFMs were activated using oxygen plasma treatment followed by amine insertion, which allows the immobilization of fibronectin through EDC/NHS chemistry. The medullary TECs presented increased proliferation, viability, and protein synthesis when cultured on fibronectin-functionalized eFMs (FN-eFMs). These cells showed a spread morphology, with increased migration toward the inner layers of FN-eFMs and the production of thymic ECM proteins, such as collagen type IV and laminin. These results suggest that FN-eFMs are an effective substrate for supporting thymic cell cultures.

Soluble antigens from the neurotropic pathogen Angiostrongylus cantonensis directly induce thymus atrophy in a mouse model.

The nematode Angiostrongylus cantonensis (A.C.) is a neurotropic pathogen; stage-III larva invade the human (non-permissive host) central nervous system (CNS) to cause eosinophilic meningitis or meningoencephalitis accompanied by immunosuppression. In an A.C.-infectedmouse (another non-permissive host) model, CNS damage-associated T cell immune deficiency and severe inflammation were proposed to result from activation of the hypothalamic-pituitary-adrenal (HPA) axis. However, glucocorticoids are anti-inflammatory agents. Additionally, while defects in thymic stromal/epithelial cells (TECs) are the major reason for thymic atrophy, TECs do not express the glucocorticoid receptor. Therefore, activation of the HPA axis cannot fully explain the thymic atrophy and inflammation. Using an A.C.-infected mouse model, we found that A.C.-infected mice developed severe thymic atrophy with dramatic impairments in thymocytes and TECs, particularly cortical TECs, which harbor CD4+CD8+ double-positive thymocytes. The impairments resulted from soluble antigens (sAgs) from A.C. in the thymuses of infected mice, as intrathymic injection of these sAgs into live mice and the addition of these sAgs to thymic cell culture resulted in thymic atrophy and cellular apoptosis, respectively. Therefore, in addition to an indirect effect on thymocytes through the HPA axis, our study reveals a novel mechanism by which A.C. infection in non-permissive hosts directly induces defects in both thymocytes and TECs via soluble antigens.

The definition of lymphocyte activating factor: giving a helping hand to serendipity.

The definition of lymphocyte activating factor: giving a helping hand to serendipity.

Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Proteasome inhibition with bortezomib depletes plasma cells and autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients (P5146)

Abstract In the autoimmune disease myasthenia gravis (MG), autoantibodies against the muscle AChR are mainly produced by both short- and long-lived plasma cells, which are resistant to standard immunosuppressive drugs (i.e. glucocorticoids). A novel therapy to eliminate plasma cells is the proteasome inhibitor bortezomib, which is used to treat patients with multiple myeloma (MM, a plasma cell malignancy). Previously, we demonstrated that bortezomib also reduced autoantibody titers in an animal model of MG (Gomez, A. M. J. Immunol. 2011). The thymus of MG patients is frequently enriched in germinal centers and contains plasma cells that produce autoantibodies in vitro, even after irradiation (which depletes B and T lymphocytes). We studied the in vitro effects of bortezomib in cultured thymus cells from MG patients undergoing therapeutic thymectomy. Treatment with a single dose of bortezomib blocked the production of these pathogenic autoantibodies, reduced the total IgG levels and eliminated plasma cells. Ultrastructural signs of apoptosis were detected in plasma cells as early as 8 h after addition of bortezomib; at 24 h, no plasma cells could be detected. Moreover, we demonstrated that the minimum concentration of bortezomib that eliminates plasma cells in vitro is 60-fold lower than the peak concentration found in MM patients treated with bortezomib, suggesting that low doses might be effective for eliminating plasma cells in patients with antibody-mediated autoimmune diseases.

Investigation on Thermophysical Properties of Thymic Cell Cultures Exposed to Electromagnetic Fields

Normal 0 7.8 ? 0 2 false false false EN-US ZH-CN X-NONE MicrosoftInternetExplorer4 The paper deals with an investigation of the possible effects of RF electromagnetic fields towards the environment and human health. An experimental facility was designed and built and in vitro experiments were carried out on thymic cell cultures. They were exposed for three and six hours to 144 MHz RF electromagnetic fields. The effects were analysed in terms of the possible variation of their thermophysical properties (density, viscosity, specific heat). Results showed that the tested configuration does not induce alterations of the thermophysical properties of the tested cell cultures.

Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice

Several studies have characterized exosomes derived from different cell sources. In this work we set the goal of proteomic characterization of two less studied populations of membrane vesicles, microvesicles (100-800 nm) and apoptotic bodies (> 800 nm) released by thymus cells of BALB/c mice. The vesicles were isolated by the combination of differential centrifugation and gravity driven multistep filtration of the supernatant of thymus cell cultures. The size distribution of vesicle preparations was determined by transmission electron microscopy. Proteins were released from the vesicles, digested in solution, and analyzed using nano-HPLC/MS(MS). Ingenuity pathway analysis was used to identify functions related to membrane vesicle proteins. In apoptotic bodies and microvesicles we have identified 142 and 195 proteins, respectively. A striking overlap was detected between the proteomic compositions of the two subcellular structures as 108 proteins were detected in both preparations. Identified proteins included autoantigens implicated in human autoimmune diseases, key regulators of T-cell activation, molecules involved in known immune functions or in leukocyte rolling and transendothelial transmigration. The presence and abundance of proteins with high immunological relevance within thymocyte-derived apoptotic bodies and microvesicles raise the possibility that these subcellular structures may substantially modulate T-cell maturation processes within the thymus.

Abnormal increase in multinuclear macrophages in primary cultures of BUF/Mna rat thymomas.

The in vitro characteristics of spontaneous thymomas from BUF/Mna rats, a strain with a high incidence of these tumors, were studied. In primary cultures, the adherent cells consisted of mononuclear macrophages, mono- and multi-nuclear epithelial cells and some fibroblastic cells on day 3. The macrophages rapidly increased in number with the formation of large multinuclear cells by day 9. A modest increase in the number and nuclearity of macrophages was also noted in adherent cultures of normal thymuses from 5-week-old BUF/Mna rats. On the other hand, in cultures of thymic cells from 1-year-old or 5-week-old ACI/NMs rats, a normal control rat strain, macrophages did not increase in number and only rarely formed multinuclear cells in adherent cell cultures. These results suggest that abnormal proliferation signal(s) to thymic macrophages and/or their progenitor cells accompanies and may be involved in the development of thymomas in BUF/Mna rats.

Stimulatory effect of thymic factor(s) on steroidogenesis in cultured rat granulosa cells

Thymic cells from immature female rats were isolated and used for production of thymic cell culture conditioned medium (TCM). Granulosa cells were obtained from immature diethylstilbestrol (DES)-treated rats. TCM stimulated basal progesterone and estradiol secretion from the granulosa cells in a dose and time dependent manner. Maximal stimulation of progesterone production occured at 48 hours of incubation, during which period TCM caused approximately 5 times more progesterone secretion than heart cell conditioned medium (HCM) or mock extract (ME). The maximum progesterone secretion by granulosa cells occured when they were exposed to 48% TCM causing 7 times more progesterone secretion than controls. Under the same maximum stimulatory conditions, however, TCM only approximately doubled estradiol secretion compared to concentrations secreted in the presence of HCM or ME. Thus, the effect of TCM on progesterone secretion was more prominent than its effect on estradiol secretion. The stimulatory action of TCM was not mimicked by HCM, thymosin-α 1 or thymulin. Furthermore, the stimulatory action of TCM on steroidogenesis did not appear to be mediated by the cAMP system. The stimulatory factor(s) in TCM were heat, acid and acetone labile, but could not be sedimented by activated charcoal. Thus, the present study demonstrates that the secretory product(s) of thymic epithelial cells can stimulate steroidogenesis in cultured rat granulosa cells. Our data imply that thymic factor(s) may have a direct effect on ovarian function.

Stimulation of thymus cells by the calcium ionophore A23187 in the presence of LPS and 2-ME: on the mode of action of A23187 and 2-ME

The stimulation of murine spleen and thymus cells by the calcium ionophore A23187 was studied using a serum-free culture technique. In cultures of spleen cells, rabbit IgG was capable of replacing A23187 in bringing about the stimulation of cells. Low concentrations of A23187 were inhibitory to proliferating B lymphocytes. In cultures of thymus cells a stimulating activity of A23187 was observed only if LPS was present in the medium.

Anti-acetylcholine receptor antibody specificities in serum and in thymic cell culture supernatants from myasthenia gravis patients.

We investigated the role of the thymus in myasthenia gravis by comparing the antigenic specificities of anti-acetylcholine receptor antibodies (anti-AChR), defined by competition with mouse monoclonal antibodies that bind to five different regions on human muscle AChR, in thymic culture supernatants and in serum pre- and post-thymectomy. Anti-AChR specificities present in the serum were broadly unchanged in 16 non-thymoma and six thymoma patients 7-30 months after thymectomy compared with an initial sample, although total anti-AChR frequently fell. The fine specificities of the anti-AChR synthesized in vitro by cultured lymphocytes from the thymus of ten patients (without thymoma) correlated significantly with that of the anti-AChR in the serum at the same time. We conclude that AChR-specific B cells in the thymus are representative of the total AChR-specific repertoire, and that thymectomy does not selectively deplete particular B cell clones.

Cells involved in the immune response: XXXI. The role of the spleen in the primary and secondary immune responses in the normal adult outbred rabbit: The initial localization of memory cells to the spleen and their subsequent dissemination to the thymus and peripheral lymph nodes

Normal adult outbred rabbits were immunized intravenously (iv) with sheep erythrocytes (SRBC). At varying times thereafter, the different lymphoid organs were investigated for spontaneous and culture-induced antibody secreting cells by the aqueous hemolytic plaque-forming cell (PFC) technique. During the phase of active antibody formation (Days 3 to 30), immediate PFC, indicative of spontaneous antibody synthesis and secretion, were detected principally in the spleen. In the early postimmune memory period (Days 30 to 90), memory cells capable of generating PFC following secondary immunization in in vitro culture with SRBC were detected only in the spleen. However, by 4 months postimmunization, memory cells were detected in the thymus and popliteal lymph node (PLN) as well as in the spleen. The number of memory cells in the thymus and PLN was significantly higher by 6 months postprimary iv immunization and was even further elevated by 9 months postprimary iv immunization. Following in vivo secondary immunization by the iv injection of SRBC 2 or 6 months postprimary immunization, immediate PFC were detected in large numbers in the spleen, the bone marrow, and the blood, marginally in the PLN and not at all in the thymus. Similar results were obtained at 9 months following primary immunization with SRBC with the exception that large numbers of immediate PFC were detected in the PLN following secondary iv immunization. Following culture of these lymphoid cells for 5 days in vitro with SRBC, the thymus and PLN cells, as well as the spleen cells, generated large numbers of PFC. Since immediate PFC were never detected among the freshly isolated thymus cells whereas thymic cell cultures 6 and 9 months postprimary iv immunization invariably generated large numbers of PFC following secondary immunization in vitro, the thymus memory cells would appear to be inaccessible to particulate antigen injected intravenously; they can only be detected following activation by the antigen in culture. The PFC generated by thymus memory cells (and spleen and PLN) were totally inhibited by the inclusion of sheep anti-rabbit IgG into the PFC assay. This finding demonstrates unequivocally that the plaques induced by thymus cells, just as the plaques induced by spleen and PLN cells, are antibody mediated and not false plaques. Therefore, the thymic PFC cells must be antibody-secreting B-memory cells since T cells do not synthesize or secrete immunoglobulins.

Interleukin-2 production in the neonatal mouse.

Neonatal mouse spleen and thymus cells fail to produce interleukin-2 (IL-2) in vitro. In vitro IL-2 production by spleen cells begins to develop between 20 and 27 days of age and reaches adult levels at 40 days of age. Thymus cells do not develop the ability to produce IL-2 in vitro. Addition of neonatal spleen, neonatal thymus, or adult thymus cells to adult spleen cell cultures inhibits IL-2 production. Treatment of the added cells with mitomycin does not abrogate their suppressor activity. Soluble factors, obtained from three-day cell cultures of neonatal spleen, neonatal thymus, or adult thymus also suppress IL-2 production in adult spleen cell cultures. Addition of interleukin-1 raises IL-2 production in adult thymus, neonatal spleen, or neonatal thymus cell cultures slightly, but the IL-2 productivity is still considerably lower than that of adult spleen in the absence of IL-1. The results indicate that suppressor cells for IL-2 production are present in neonatal spleen and thymus. The splenic suppressor cell activity disappears after birth, but thymic suppressor cell activity is retained into adulthood.

A simple rapid method for detection of IL-2 in a physiological medium

The Con A-activated proliferation of C57BL/6 mice thymus cell suspension is completely blocked by a low concentration of hydrocortisone. This inhibition is reversed by the addition of a 24 h Con A-activated splenic cell supernatant to the thymic cell culture. The restoring activity is completely destroyed by heating. Restoration is not by addition of IL-1-rich supernatant from an LPS-activated peritoneal adherent cell population. The supernatant of a 24 h splenic cell culture activated by Con A in the presence of indomethacin not only restores hydrocortisone-blocked themocyte reactivity but also enhances thymic cell stimulation. Addition of PGE to the splenic cell culture inhibits the restoring potency of the supernatant. These results are in accord with the proposition that IL-2 is responsible for the restoring phenomenon. These properties have been used to develop a semi-quantitative method for detection of IL-2 in a physiological medium.

Activating effects of interferons, lymphokines and viruses on cultured chicken macrophages1

Cultured chicken bone-marrow-derived and blood monocyte-derived macrophages could be activated by various treatments: (1) With crude lymphokine preparations obtained from Concanavalin A (ConA)-stimulated chicken spleen or thymus cell cultures: (2) with virus-induced interferons (IFN) from cultured chicken embryo fibroblasts or macrophages; (3) with inactivated reovirus or live infectious bursal disease virus (IBDV). Macrophage activity was expressed by cytostatic effects against lymphoblastoid target cells, and by morphological changes, such as enhanced spreading of the cells. The macrophage-activating capacity of lymphokine preparations (50% cytostasis-inducing dose) was closely associated with their antiviral activity (IFN units). According to its physico-chemical properties, ConA-induced lymphocyte interferon was considered to be IFN-gamma, but acid-and heat-resistant IFN-alpha or IFN-beta may also have been present in spleen cell or thymocyte culture supernatants. Virus-induced interferons (IFN-alpha/beta) were less effective in macrophage activation than in antiviral activity. Experimental results strongly suggested that macrophage activation by viruses was mediated by endogenous IFN.

Differentiation block of prethymic lymphocytes during Moloney-virus-induced lymphoma development associated with a thymic epithelial defect.

Previous cytokinetic studies in Moloney-virus (M-MuLV)-induced lymphomas of BALB/c mice showed an intrathymic maturation block of prethymic lymphocytes derived from hematopoietic tissues. Thymus-cell cultures during the latent period of lymphoma development showed a proportion of nonlymphoid cells (NLC) from uninfected mice of 0.1% (3 days) to 0.002% (20 days), rising in infected mice to 1-2% after 5 weeks. Concomitantly, thymic epithelial cells exhibit progressive degenerative changes in vivo in infected mice with virus replication and in vitro a marked cellular polymorphism with nuclear atypia becomes overt. Immunofluorescence studies of thymopoietin II and serum thymus factor in epithelial cells indicate a marked decrease of these hormones in the epithelial cells from infected mice. These results suggest a functional defect of virus-infected thymic epithelial cells which causes a progressive accumulation of nondifferentiating T-cell precursors.

Induction of interferon-γ in mouse spleen cells by OK-432, a preparation of Streptococcus pyogenes

A bacterial antitumor and immunopotentiating agent, OK-432, induced Interferon in the spleen cell cultures but not in the thymus cell cultures of various inbred strains of mice. When 1 × 10 7 spleen cells were cultured in the presence of 5 μg/ml of OK-432, interferon activity was detected as early as 4 hr later and reached a maximum level of about 160 to 500 units/ ml 24 hr later. OK-432-induced interferon was mainly an IFN-γ of molecular weight approximately 40,000, but also contained IFN-α and IFN-β.

Lympho-stromal interactions in the thymus: medullary thymocytes react with I-A determinants on autochthonous thymic stimulator cells.

AbstractPrimary (1o) and secondary (2o) syngeneic mixed lymphocyte culture (SMLC) reactions in autochthonous murine thymus cell cultures (peanut agglutinin‐unreactive) or syngeneic mixed thymus‐spleen cell cultures were studied. The 1o thymic SMLC responder cells were Thy‐1+, Lyt‐1+, Lyt‐2−. The 1o thymic syngeneic mixed lymphocyte reaction (SMLR) could be inhibited with monoclonal antibodies directed against determinants Ia.m1 and Ia.m6, in the absence of complement.Positive selection of thymic SMLC cells from 1o cultures was achieved by enriching reactive lymphoblasts on discontinuous Percoll density gradients. The 2o memory populations were obtained by allowing the selected lymphoblasts to revert back to small resting 2o cells in the absence of SMLC stimulation. The thymic 2o SMLC populations were highly enriched in their activity and were almost exclusively specific for self stimulator cells.The 2o thymic SMLC cells were found to be Thy‐1+, membrane immunoglobulin‐negative, H‐2K/D+, I‐A−, I‐E− and I‐J−. They were hardly lysed by anti‐Lyt‐1 antibodies and not at all lysed by monoclonal antibodies detecting Qat‐4 and 5, and Lyt‐2 antigens, in the presence of complement. Stimulator cells were found in fast‐sedimenting or low‐density fractions of thymus. They expressed H‐2K/D, I‐A‐, and I‐E antigens but lacked membrane immunoglobulin and Thy‐1. Not all I region determinants seemed to be of equal importance in the 2o thymic SMLC: I‐A subregion compatibility between responding and stimulating cells was a minimal requirement for the induction of 2o thymic SMLC responses. Although several relevant anti I‐region antibodies reacted with the stimulator cells in the presence of C, these and other anti‐Ia antibodies varied considerably in their capacity to block 1o and 2o autochthonous thymic SMLR in the absence of complement.Our finding that the thymus contains the responder as well as the stimulator cells capable of interacting in an autochthonous proliferative reaction will be discussed with regard to its implications for the intrathymic differentiation of T lymphocytes.

T cell hybridoma-derived regulatory factors. I. Production of T cell growth factor following stimulation by concanavalin A.

A cloned T cell hybridoma (123) was shown to produce T cell growth factor (TCGF). Supernatants of cultures of hybridoma-123 that had been stimulated with concanavalin A caused T cell blasts or a cloned T cell line to proliferate, allowed a mitogenic response to concanavalin A by thymocytes and by lymph-node cells depleted of accessory cells by treatment with anti-Ia serum and complement, and permitted the generation of both polyclonal and antigen-stimulated cytotoxic T lymphocytes in cultures of thymus cells. These observations suggest that the hybridoma is producing a factor with an identical spectrum of activities to that associated with TCGF derived from mitogen-stimulated spleen cells and indicate that the T cell is the source of TCGF. Together with other evidence that the same hybridoma produces activity affecting B lymphocytes, myeloid progenitor cells, and pluripotential stem cells, these experiments confirm the role of the activated T cell as a regulator of hemopoietic and lymphoid systems. T cell hybridomas should prove invaluable for biochemical and genetic analysis of these factors.

A B Lymphocyte Mitogen Extracted from a Fungus Peziza vesiculosa1

A water-soluble mitogen was extracted with hot-water from the fruiting bodies of a fungus, Peziza vesiculosa, collected in the wild. The active substance, named vesiculogen, was able to stimulate selectively murine B cells because mitogenic activity was observed in the spleen cell cultures of congenitally athymic nude mice, but not in the thymus cell cultures. The possibility that the mitogenicity of vesiculogen was due to lipopolysaccharide was denied completely by the following evidence: 1) lipopolysaccharide in vesiculogen was undetectable(less than 0.001% in the Limulus test), 2) vesiculogen was able to stimulate strongly DNA synthesis of spleen cells from C3H/HeJ mice, and 3) the mitogenic activity of vesiculogen was not inhibited by polymyxin B. Vesiculogen increased antigen-nonspecifically the number of direct plaque forming cells to sheep erythrocytes, horse erythrocytes, and trinitrophenylated-horse erythrocytes. This result shows that vesiculogen acts as a polyclonal B cell activator on murine spleen cells.