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Thymic B cells represent a numerically minor cell population located primarily at the cortico-medullary junction. Their biological role is unclear. B cell-deficient μMT mice exhibited reduced medullary thymic epithelial cell (mTEC) numbers and reduced MOG and insulin mRNA expression. Lymphotoxin produced by B cells was critical for normal tissue restricted antigen (TRA) expression, suggesting that B cells regulate self-antigens through their production of LT. These results reveal an unexpected role of B cells in mTEC maintenance and expression of TRAs through their production of LT.

Although multiple sclerosis (MS) is considered primarily as a demyelinating disease, neuronal damage is abundant and correlates with the neurological deficit. Therefore, we investigated the frequency and characteristics of human T cells specific for synapsin-a neuronal protein highly conserved among species. Synapsin specific T cell responses were detected at a frequency similar to that of MBP specific T cells in MS patients, one patient with acute demyelinating encephalomyelitis (ADEM) and controls. Long-term T cell lines specific for synapsin exhibited a CD3(+), CD4(+), CD8(-) phenotype and produced high amounts of tumor-necrosis-factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) after antigen specific stimulation, whereas lymphotoxin (LT), interleukin-4 (IL-4) and interleukin-10 (IL-10) were detectable in smaller quantities.

The anaphylatoxin C5a is a potent mediator of inflammation in the CNS. We analyzed the intracerebral expression of the C5a receptor (C5aR) in a model of closed head injury (CHI) in mice. Up-regulation of C5aR mRNA and protein expression was observed mainly on neurons in sham-operated and head-injured wild-type mice at 24 h. In contrast, in TNF/lymphotoxin-alpha knockout mice, the intracerebral C5aR expression remained at low constitutive levels after sham operation, whereas it strongly increased in response to trauma between 24 and 72 h. Interestingly, by 7 days after CHI, the intrathecal C5aR expression was clearly attenuated in the knockout animals. These data show that the posttraumatic neuronal expression of the C5aR is, at least in part, regulated by TNF and lymphotoxin-alpha at 7 days after trauma.

The aim of this study was to analyze associations between myasthenia gravis (MG) and polymorphisms in the tumor necrosis factor (TNF) region in 79 Swedish patients and 155 unrelated controls. The frequency of the TNFa2 allele of a microsatellite located 3.5 kb upstream of the lymphotoxin alpha (LT- α ) gene in the TNF region was found to be increased in overall MG patients compared to controls. The frequency of the short 5.5 kb fragment (TNFB*1) of a bi-allelic Nco I RFLP polymorphism located at the first intron of the LT- α gene was increased in patients with an early onset of disease compared to patients with a later onset.

In experimental allergic encephalomyelitis (EAE) myelin basic protein (MBP) specific T cells differ in their encephalitogenic potential. To investigate the functional diversity of human MBP specific T cell lines, we analysed their cytotoxic activity against human astrocytes and monocytes. Five out of 14 MBP specific T cell lines killed astrocytes in the presence of MBP. Nevertheless, all lines lysed blood derived monocytes. T cell lines that lysed astrocytes efficiently in the presence of MBP, recognized peptide aa 80-99/86-105 in context with HLA-DRB5 * 0101, peptide aa 50-69/61-83 in context with HLA-DRB1 * 1501 and peptides aa 139-153, and aa 148-162 in context with HLA-DRB1 * 0101. There was no correlation of MBP-mediated lysis of astrocytes with TCR-Vbeta usage, HLA-restriction and the production of tumor-necrosis-factor-alpha (TNF-alpha), lymphotoxin (LT) and interferon-gamma (IFN-gamma). Different lysis of astrocytes, however, revealed a functional heterogeneity of MBP specific T cells, which was not observed by using monocytes as targets.

The cytokine lymphotoxin (LT) is known to exist in two forms, secreted LT α and a membrane-bound LT α / β complex. LT α shares the same receptor as tumor necrosis factor α and LT β is recognized by its receptor, LT β R. Since LT has been associated with oligodendrocyte pathology, the present study has examined the expression of these molecules by immunocytochemistry in diseased and normal CNS tissue, with a panel of monoclonal antibodies (mAb) to LT α , LT β and LT β R. Of three mAb to LT β , two (B27 and C37) gave specific membrane staining on astrocytes, as well as lymphocytes. The third anti-LT β mAb, B9, was selectively immunoreactive for oligodendrocytes, suggesting specific recognition sites. The reactivity was not specific for multiple sclerosis (MS) since oligodendrocytes in normal and non-MS CNS tissue also displayed positivity. MAb to LT β R reacted with astrocytes only, giving a punctate membrane staining pattern suggestive of receptor sites. MAb to LT α gave strong reactivity on lymphocytes in active MS lesions and weak reactivity on microglia within lesion areas. These results show that mAb to LT α and LT β recognize different cell types within the CNS. Furthermore, individual mAb against LT β were capable of distinguishing between astrocytes and oligodendrocytes, perhaps indicative of different epitopes on LT β . The presence of LT β R on astrocytes suggests possible interactions between infiltrating lymphocytes and astrocytes via the LT pathway.

Myasthenia gravis (MG) is a neuromuscular disorder mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) on the postsynaptic membrane of the neuromuscular junction. The production of antibodies is regulated by T cells by means of immunoregulatory cytokines. To investigate the involvement of TNF-alpha, lymphotoxin (LT), IL-6, IL-10, IL-12 and perforin in MG, numbers of cytokine mRNA expressing blood mononuclear cells (MNC) were examined in patients with MG and controls. LT belongs to the Th1 cell related cytokines, IL-6 and IL-10 to the Th2 type, TNF-alpha is produced by both Th1 and Th2 cells, IL-12 induces T cell switch towards the Th1 type and perforin is an effector molecule inducing cell lysis. Short term culture of MNC with AChR revealed augmented levels of AChR-reactive TNF-alpha, LT, IL-6, IL-10, IL-12 and perforin mRNA expressing cells in MG compared to levels obtained without AChR or in presence of control antigen. AChR-reactive TNF-alpha, IL-6, IL-10, IL-12 and perforin mRNA expressing cells were higher in MG than controls. These findings suggest that the cytokines TNF-alpha, LT, IL-6, IL-10 and IL-12, and the cytolytic effector molecule perforin are also involved in MG.

A possible correlation between the pathogenicity of autoimmune T cells and their lymphokine production, expression of functional adhesion molecules and expression of some surface antigens was examined. We used four retinal antigen-specific Lewis rat T cell lines and sublines: one specific to the major pathogenic epitope of the human retinal soluble antigen (S-Ag; residues 337-356), and three specific to the major pathogenic epitope of the bovine interphotoreceptor retinoid binding protein (IRBP; residues 1177-1191). The lines have different degrees of uveitogenicity, from highly pathogenic to nonpathogenic. All four T cell lines produced roughly equivalent amounts of interferon-gamma, lymphotoxin/tumor necrosis factor (TNF alpha/beta), interleukin-3, interleukin-6 and transforming growth factor-beta. Interleukin-4 activity could not be detected. The lines also expressed similar levels of functional adhesion molecules, as measured by binding to cultured rat aorta endothelial cells. The nonpathogenic subline, however, was the lowest responder to antigenic stimulation with respect to proliferation and interleukin-2 production. Examination of cell surface antigens showed that in contrast to the other lines, the majority of cells in the nonpathogenic subline lacked detectable expression of CD4. No difference was found in the level of expression of the IL-2 receptor and T cell antigen receptor among the four lines. Because CD4 is the restricting element in these lines, reduced CD4 expression in the nonpathogenic subline may at least partially explain its poor response in vitro to antigenic stimulation. All three attributes could be connected to lack of pathogenicity of this line in vivo. These results support the contention that class II-restricted recognition of autoantigen within the neuroretina by uveitogenic T lymphocytes must occur as an initial step in the pathogenesis of EAU. A defect in this step will preclude pathogenesis regardless of some other functional attributes possessed by effector T cells, such as production of inflammatory lymphokines and expression of adhesion molecules.

Supernates from myelin basic protein (MBP)-reactive T cell lines have been tested by a battery of assays for cytotoxicity against oligodendrocytes in vitro. All supernates tested demonstrated cytotoxic activity in both a dose- and time-dependent manner that ranged from 29.1% to 55.8% after 48 h incubation. Oligodendrocyte cytotoxicity mediated by MBP-reactive T cell lines was not antigen specific since lines reactive with purified protein derivative (PPD) of tuberculin showed similar cytotoxicity. Supernates cytotoxic to oligodendrocytes were equally effective against syngeneic and non-syngeneic target cells, but had no effect upon astrocytes. Neutralization studies revealed that oligodendrocyte cytotoxicity mediated by MBP-reactive T cell supernates could only be partially attributed to lymphotoxin/tumor necrosis factor activity, and was not associated with perforin, serine proteinase or N-methyl-D-aspartate (NMDA) receptor agonists.

A complete prevention of clinical experimental autoimmune myasthenia gravis (EAMG) was observed in lymphotoxin (LT)-alpha deficient (LT-alpha(-/-)) mice compared to LT-alpha(+/+) mice when immunized with acetylcholine receptor. However, only a partial prevention of clinical EAMG incidence was observed in LT-beta(-/-) mice compared to LT-beta(+/+) mice. LT-alpha(-/-)and LT-beta(-/-) mice had lower mean titers of total IgG, IgG(1), IgG(2a) and IgG(2b) and higher or equal mean titers of IgM anti-AChR antibodies compared to controls. Therefore, LT-alpha(-/-)and LT-beta(-/-) AChR immunized mice are capable of mounting a primary (IgM) humoral immune response to AChR, but are less capable of switching to the pathogenic anti-AChR IgG isotypes. LT could play a significant role in the pathogenesis of myasthenia gravis.

We previously reported that the CD4+ suppressor cells (Ts) that regulate recovery of Lewis rats from experimental autoimmune encephalomyelitis (EAE) produce transforming growth factor-beta (TGF-beta). We also reported that TGF-beta downregulates interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2) production, by the CD4+ effector T cells (Te) that mediate EAE. We now report that TGF-beta also inhibits the production of tumor necrosis factor/lymphotoxin (TNF/LT) by EAE effector cells. When activated in vitro with myelin basic protein (MBP), Te produced TNF/LT, as measured using a WEHI 164 cytotoxicity assay. The specificity of cytokine action was demonstrated using neutralizing antibodies to TNF/LT. When added to the Te+MBP cultures, TGF-beta inhibited TNF/LT production in a dose-dependent fashion. Moreover, neutralizing anti-TGF-beta antibodies augmented TNF/LT production in the Te+MBP cultures. We also confirm that TGF-beta inhibits adoptive transfer of EAE. In contrast, murine IL-10 only partially inhibited TNF/LT and IFN-gamma production by Te. We conclude that TGF-beta production by Ts plays a major role in recovery from EAE in the Lewis rat by inhibiting TNF/LT and IFN-gamma production by the effector cells that mediate EAE.

Among the myelin basic protein (MBP)-specific T-cell clones mediating experimental allergic encephalomyelitis (EAE), which were established from SJL/J mice, one clone was found to have lost its encephalitogenicity during long-term passages in vitro, altoough the clone keeps its specific reactivity to the encephalitogenic determinant lying in the sequence of guinea pig MBP 89–101. To clarify the difference between the encephalitogenic T-cell clone (4b.14a) and non-encephalitogenic T-cell clone (4b.14a/n), we examined various lymphokines secreted into the culture media of 4b.14a and 4b.14a/n. The results show that the activities of lymphotoxin, interferon-γ or interleukin-2 were not different between encephalitogenic clones and 4b.14a/n, whereas the activity of tumor necrosis factor-α, possibly secreted from antigen-presenting cells, was higher in culture media of 4b.14a/n. Moreover, the culture fluid of both 4b.14a/n and 4b.14a revealed suppressive effect on the proliferation of 4b.14a stimulated by MBP 89–101, but the effect was not different between the two clones. Thus, it is suggested that neither production of lymphokines examined so far nor soluble suppressive substance is related to the loss of encephalitogenicity of the T-cell clone.

Myelin basic protein (MBP)-specific T cell lines derived from SJL mice lose the ability to transfer adoptively experimental allergic encephalomyelitis (EAE) after 5–6 restimulations with antigen in vitro. In order to test whether such lines were suppressive, non-encephalitogenic T cell lines were co-cultured with a freshly derived encephalitogenic T cell line. Following co-culture in the presence of MBP and irradiated syngeneic spleen cells the mixture was tranferred adoptively to syngenic recipients. Severe EA was observed in recipients of the encephalitogenic cell line alone but not in animals which received the co-culture. A co-culture period was required as mixing the encephalitogenic and non-encephalitogenic T cell lines just prior to transfer was without effect. Not all non-encephalitogenic cell lines were found to be suppressive. Culture fluids from the suppressive, but not the non-suppressive lines were found to inhibit MBP-driven proliferation of T cell clones and encephalitogenic lines in vitro. Nineteen of 55 MBP-specific T cell clones derived from suppressive lines were found to elaborate the supppressive supernatant activity. The suppressive effect was not antigen-specific since the same culture supernatants inhibited proliferation of an ovalbumin-specific SJL T cell clone. The suppressive effect became apparent only after T cell lines had lost encephalitogenicity and was not mediated by tumor necrosis factor, lymphotoxin or prostaglandin.

Growth of cultured human glioblastoma cells was profoundly inhibited by concentrated lymphokines prepared from mitogen-activated blood mononuclear cells of normal donors. Cloning efficiency of glioma cells and their absolute number were decreased as well. Partially purified leukoregulin, free of lymphotoxin, tumor necrosis factor and γ-interferon, similarly suppressed DNA synthesis and clonogenicity. The decrease in absolute numbers of tumor target cells indicated that leukoregulin was directly cytolytic as well as cytostatic for human glioblastoma cells. Our data indicate that leukoregulin is at least one of the factors produced by activated lymphocytes which inhibits the proliferation of human glioblastoma in vitro.