Novel Tcr Tcra-V and -J transcripts expressed in rat myelin-specific T-cell lines.
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Amino Acid Sequence↗
Biomedical subjects
H Wekerle
Publications and source records attributed to H Wekerle.
New approaches to the study of T cell autoimmunity in multiple sclerosis. Meeting report on the 2nd Workshop on T cell Autoimmunity in MS, Perugia, Italy, 26-28 November, 1992.
This report discusses some of the papers presented during the 2nd T Cell Autoimmunity in MS Workshop held in Perugia on November 26-28, 1992. The 'trait d'union' of the meeting was the awareness that recent studies have failed to uncover clear-cut differences in T cell reactivity to autoantigens between patients with multiple sclerosis and healthy individuals. Hence, new strategies have been proposed to try to distinguish between 'pathological' autoimmunity in patients and 'natural' autoimmunity in healthy individuals. Furthermore, the studies have been extended to new potential autoantigens (myelin constituents other than myelin basic protein, superantigens, heat shock proteins) and new susceptibility genes such as those coding for transport-associated proteins (Tap-1 and Tap-2).
Autoimmune Diseases↗
Experimental autoimmune encephalomyelitis as a model of immune-mediated CNS disease.
Experimental autoimmune encephalomyelitis models are used to analyze the generation and organization of the myelin-specific autoimmune repertoire, and potential immunoregulatory loops preventing spontaneous activation of encephalitogenic T cells. These lymphocytes are profoundly modulated by infectious agents, which may trigger, or more commonly, prevent experimental autoimmune encephalomyelitis. The development and resolution of the pathogenic central nervous system infiltrations is controlled by locally produced cytokines that cause recruitment of infiltrate cells, and their disappearance. Several of the new findings seem now to be applicable for therapeutic strategies, especially with the aim of interfering with immunospecific recognition steps involved in disease generation.
Animals↗
T-cell autoimmunity in the central nervous system.
Studies of myelin basic protein (MBP)-specific T lymphocytes have been extraordinarily informative for several distinct aspects of (neuro) immunology. They indicated for the first time that myelin directed central nervous system (CNS) inflammation is mediated by specific autoaggressive T cells. This provided a conceptual basis for understanding the pathogenesis of anti-myelin autoimmunity in general. We assume at present that human multiple sclerosis (MS) is also caused by myelin-specific autoaggressive T cells, and that these T cells create the inflammatory changes typical for the generation of a mature demyelinating plaque. Large scale demyelination seems, however, not to be caused by purely cellular mechanisms. Second, studies using MBP-specific T-cell lines have critically contributed to elucidating the immune status of the CNS. They showed that the CNS is not completely secluded from the circulating immune cells. Activated, though not resting lymphocytes can pass through the endothelial blood-brain barrier and migrate through the brain tissues. There, in the CNS, some glia components, microglia and astrocytes can be induced by T-cell cytokines to act as efficient antigen-presenting cells. Thus, the CNS is subject to immune surveillance, though to a specialized, adapted variant thereof. Finally, studies using MBP-specific T cells established for the first time the presence of potentially autoaggressive T cells within the normal immune repertoire. This implies counterregulatory regulation required to keep the autoreactive T cells in a safe state of rest. Most recently, the analysis of the intrathymic anti-MBP T-cell repertoire may change our views on the generation of myelin-specific self tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.
Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.
Animals↗
Specific immune complexes augment in vitro acetylcholine receptor-specific T-cell proliferation.
We investigated the interaction between acetylcholine receptor (AChR)-specific T-helper cells from patients with myasthenia gravis and murine monoclonal anti-AChR antibodies. At optimal antigen concentration, anti-AChR antibodies neither enhanced nor impaired T-cell responses. However, at substimulatory antigen concentration, addition of anti-AChR antibodies substantially enhanced the proliferation of AChR-specific T cells. In spite of low amounts of antigen, immune complex formation allowed highly efficient capture and uptake of antigen via Fc receptors on antigen-presenting cells, which could be inhibited by an antibody to Fc receptors. Immune complex-mediated stimulation of sensitized AChR-specific T lymphocytes in vivo may contribute to the exacerbation of the disease, and demonstrates the interaction between T and B lymphocytes in myasthenia gravis.
Adjuvants, Immunologic↗
Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell death as a mechanism to control inflammation in the brain.
In experimental autoimmune encephalomyelitis (EAE) myelin-specific T lymphocytes attack the myelinated tissue of the central nervous system (CNS). In the Lewis rat, EAE as a rule has an acute, monophasic course. With spontaneous clinical recovery the inflammatory CNS infiltrates are cleared from the nervous tissue within a few days. This is well in line with the remarkably low incidence of myelin-specific T cells present in EAE infiltrate. Combining immunocytochemical techniques, ultrastructural criteria and in situ nick translation we found up to 49% of T lymphocytes in EAE lesions showing signs of apoptosis at the time of recovery from disease. Our results suggest that apoptosis of T lymphocytes may be one possible mechanism to eliminate T lymphocytes from inflammatory brain lesions.
Animals↗
Human myoblasts as antigen-presenting cells.
Human myoblasts, cultured from muscle and purified to greater than 95%, were investigated for their capacity to act as facultative APC. The myoblasts reacted with antidesmin mAb and had the capacity to fuse into multinucleated myotubes in appropriate medium. The expression of HLA class I, HLA-DR, HLA-DP, HLA-DQ, intercellular adhesion molecule-1 (ICAM-1/CD54), lymphocyte function-associated (LFA) molecules LFA-1 (CD11a/CD18), LFA-2 (CD2), and LFA-3 (CD58) was investigated by FACS analysis before and after induction for various times with human rIFN-gamma, TNF-alpha, or both. Without cytokine induction, myoblasts expressed only HLA-class I and LFA-3. IFN-gamma alone or in combination with TNF-alpha induced the expression of HLA-DR and ICAM-1 reaching a plateau after 48 h, followed by HLA-DP and even later HLA-DQ. TNF-alpha alone induced only ICAM-1. The functional capacity of myoblasts to present Ag to CD4+ T cells was investigated using autologous T cell lines specific for tuberculin, tetanus toxoid, and human myelin basic protein. Noninduced myoblasts or myoblasts treated with TNF-alpha alone could not present any of these Ag to the T cells. However, myoblasts treated with IFN-gamma induced Ag-specific proliferation. In the presence of relevant Ag, myoblasts were killed by the T cells as observed by microscopy and measured by 51Cr release. Ag-specific T cell proliferation and myoblast killing was inhibited in the presence of anti-DR mAb. These results suggest that human myoblasts may act as facultative APC during local immune reactions in muscle.
Antigen-Presenting Cells↗
Diversity of T cell receptor alpha and beta chain genes expressed by human T cells specific for similar myelin basic protein peptide/major histocompatibility complexes.
T cell receptor (TcR) alpha and beta nucleotide sequences involved in the human autoreactivity to myelin basic protein (MBP) were studied by screening cDNA libraries derived from 11 independent T lymphocyte clones (TCC) established from multiple sclerosis patients and healthy donors. The TCC with defined MBP peptide specificity and HLA-DR restriction expressed multiple TcR. Even TCC recognizing the same human MBP peptide [amino acids (aa) 139-153] in identical or very similar HLA-DR context expressed diverse TcR. Two TCC which recognized peptide aa 139-153 equally well in the context of both HLA-DR2a and -DR1 molecules used distinct TcR alpha but identical beta chains. The knowledge of TcR beta and TcR alpha chain sequences of human MBP-specific T cells will allow studies correlating structure and function of TcR and their targets in MBP autoreactivity. This may have an impact on the development of immunotherapies in multiple sclerosis.
Amino Acid Sequence↗
Immune regulation in self tolerance: functional elimination of a self-reactive, counterregulatory CD8+ T lymphocyte circuit by neonatal transfer of encephalitogenic CD4+ T cells lines.
Transfer of encephalitogenic, CD4+ T lymphocyte lines into syngeneic adult Lewis rats not only leads to the development of experimental autoimmune encephalomyelitis (EAE), but, in addition, to the expansion of counterregulatory, CD8+ T lymphocyte clones which are able to lyse specifically the encephalitogenic T cells in vitro and to neutralize their encephalitogenic capacity in vivo. In striking contrast, in neonatal rats, which still lack myelin (autoantigens), injection of the same encephalitogenic lines neither mediates EAE, nor confers protection in later life against the myelin-specific T cells. In fact, this treatment results in the life-long functional elimination of counterregulatory, clonotypic CD8+ T lymphocytes, which cannot even be reinduced by repeated injections of the relevant CD4+ T line. These data seem to point to a self-protective T cell control mechanism which is developed within the immune system prior to, and thus independent of the appearance of the appropriate self antigen.
Animals↗
Diversity of T cell receptor alpha and beta chain genes expressed by human T cells specific for similar myelin basic protein/major histocompatibility complexes.
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Base Sequence↗
Interaction between oligodendroglia and immune cells: mitogenic effect of an oligodendrocyte precursor cell line on syngeneic T lymphocytes.
We analyzed cellular interactions between T lymphocytes and a recently established immortal glial line, L3 that retains several properties of immature oligodendrocytes (Aloisi et al., J Neurosci Res 27:16-24, 1990). L3 oligodendrocytes (L3-OL) cannot be induced to express class II antigens, nor do they specifically present antigen to syngeneic specific T lymphocyte. However, L3-OL strongly enhance the proliferation of freshly activated, interleukin-2(IL-2)-dependent T-line lymphocytes and concanavalin A (ConA)-activated lymphoblasts, irrespective of their antigen specificity or surface phenotype (CD4+ or CD8+). Resting and some activated T cells were susceptible to the mitogenic effect of L3-OL only in the presence of exogenous IL-2, not of other cytokines. The mitogenic effect of L3-OL did not depend on cell viability. It was observed in paraformaldehyde-fixed L3-OL cells and in membrane preparations, but not in culture supernatant. Neither intact L3-OL cells nor membrane preparations had direct IL-2 activity. The conclusion that the mitogenic effect of L3-OL cells is exerted by membrane structures acting as a costimulatory factor(s) of IL-2 is supported by the finding that it is largely blocked by a monoclonal anti-IL-2 receptor antibody. The effect is distinct from membrane-bound IL-1, membrane-bound tumor necrosis factor-alpha (TNF-alpha), IL-3, or IL-6 and cannot be reconstituted by these cytokines.
Animals↗
Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). I. Cell detachment and lysis induced by encephalitogenic CD4+ T lymphocytes.
Myelin basic protein (MBP) reactive CD4+ T lymphocytes, capable of inducing experimental autoimmune encephalomyelitis (EAE), were examined for their ability to damage target cells of central nervous system (CNS) origin. Damage was assessed by monitoring detachment of adherent astrocytes from substratum and astrocyte lysis. MBP-specific, but non-encephalitogenic CD4+ T cells mediated astrocyte detachment but not lysis. However, encephalitogenic CD4+ T cell lines were more efficient in causing astrocyte detachment and could also cause astrocyte lysis. The detachment and lytic activities of the MBP-reactive T cell lines tested were often independent of the presence of specific antigen, and were not restricted to syngeneic major histocompatibility (MHC) antigens. MBP often augmented the detaching and, if lytic, lytic activities of these T cells. The encephalitogenic CD4+ T cells also detached and lysed allogeneic 'bystander' fibroblasts in the presence of unlabelled syngeneic astrocytes, suggesting the involvement of a soluble mediator(s). Although MBP is essential for the initiation of EAE, the presence of MBP on cells of CNS origin, such as astrocytes and oligodendrocytes, does not appear to be necessary for their damage by MBP-specific CD4+ T cells. Immune CD4+ T cells, which penetrate the CNS, may disorganize brain tissue structure by lysing astrocytes directly and by damaging other brain cells indirectly by soluble mediators. Thus cellular detachment, in addition to cell lysis, mediated by MBP-specific CD4+ cells may contribute to EAE pathogenesis.
Animals↗
Protection from experimental allergic encephalomyelitis by application of a bacterial superantigen.
Certain bacterial and viral T cell stimulating proteins ('superantigens') are known to be very potent activators of T cells with certain V beta receptors. When applied in vivo these molecules induce anergy in those T cells responding to them. In this study we have investigated the influence of staphylococcal enterotoxins (SE) on myelin basic protein (MBP)-specific T cells in Lewis rats. As MBP-specific T cells in rats belong exclusively to the V beta 8.2+ CD4+ subset, the induction of experimental allergic encephalomyelitis (EAE) allows for an estimation of the functional state of the respective V beta-bearing T cells after enterotoxin-induced activation. In vitro, various MBP-specific T cell lines showed a strong selective proliferative response to staphylococcal enterotoxin E (SEE) but not to other SE. The in vitro activation by SEE induced encephalitogenic potential in these cells. After application of SEE to Lewis rats the susceptibility to induction of EAE was completely abrogated. Such SEE-treated and MBP-challenged rats did not exhibit any signs of disease and their T cells did not respond to MBP in proliferation tests. This abrogation of EAE was only found with a superantigen capable of interacting specifically with V beta 8.2+ T cells. Superantigen-mediated induction of unresponsiveness may have relevance for the analysis of pathogenetic mechanisms and for therapeutic considerations in certain T cell-mediated autoimmune diseases.
Animals↗
Transplantation of thymic autoimmune microenvironment to severe combined immunodeficiency mice. A new model of myasthenia gravis.
To study the role of the thymus in the cellular pathogenesis of myasthenia gravis (MG) we transplanted thymus tissue fragments from MG thymuses beneath the kidney capsule of severe combined immunodeficiency (SCID) mice. Immunocytochemical studies documented that the human thymus tissues are accepted as long-term grafts in the host SCID mice, with human lymphocytes, thymic stroma, and thymic myoid cells demonstrable in transplanted thymus for at least 15 weeks after transplantation. Human anti-acetylcholine receptor antibodies became detectable 1 to 2 weeks after transplantation, and in most chimeras the titers increased over at least 11 weeks to reach levels typically found in severe human MG. Human Ig deposits were detected at skeletal muscle end-plates, demonstrating that the human (auto)antibodies bound to murine acetylcholine receptor. In contrast, transfers of dissociated thymus cells only lead to a transient increase of anti-acetylcholine receptor antibodies. Our data prove that myasthenia gravis thymus is able to induce and maintain autoantibody production in immunodeprived host animals, and that this tissue contains all cellular components required for autoantibody production. Transplantation of solid thymus tissue seems to transfer an autoimmune microenvironment, which will allow direct studies of the mechanism of autosensitization inside the thymus.
Animals↗
Myelin specific, autoaggressive T cell clones in the normal immune repertoire: their nature and their regulation.
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Animals↗
Spinal cord microglia in experimental allergic neuritis. Evidence for fast and remote activation.
We have studied the response and the spatial distribution pattern of microglial cells during experimental allergic neuritis induced in the Lewis rat by the transfer of varying doses of activated T cells specific either for the P2 or P0 protein. The microglial reaction was studied immunocytochemically at the light and electron microscopic level using a panel of monoclonal antibodies which included two recently produced antibodies against rat microglial cells, Murine Clone 101 and 102. Activation of microglial cells became apparent through changes in their immunophenotype and morphology within 48 hours of T cell transfer and therefore preceded the onset of clinical disease. Activated microglial cells showed an increased expression of the complement type three receptor, the murine clone 101 and 102 determinants and major histocompatibility complex antigens. The microglial reaction in experimental allergic neuritis occurs at a site remote from the inflammatory changes in the peripheral nerve, the microglial reaction being most prominent in the dorsal and ventral grey matter of the lumbar and the thoracic spinal cord. Similar changes were also observed at this time in the terminal projection fields of the primary, afferent, sensory fibers, such as the nucleus gracilis. Subsequently, after 7 days, motoneurons, particularly in the ventral grey matter of the lumbar spinal cord, were ensheathed by perineuronal microglial cells. These perineuronal microglial cells were in close contact with the neuronal plasma membrane and occasionally appeared to detach afferent synaptic terminals from the surface. Microglial responses were not detected in animals injected with nonpathogenic T cells specific either for the purified protein derivative or ovalbumin. This early activation of microglial cells observed in experimental allergic neuritis suggests that a rapid and remote signaling might be operating in the microglial responses during T cell-mediated autoimmune diseases.
Animals↗
CD4-CD8- splenic T cells from Lewis rats recovered from experimental autoimmune encephalomyelitis respond to encephalitogenic T cells that mediate this disorder.
We previously demonstrated that encephalitogenic CD4+ T lymphocytes from the long-term cultured line S1, specific for myelin basic protein, induce a CD8+ T cell population in vivo that protects naive Lewis rats against experimental autoimmune encephalomyelitis caused by S1 cells. In order to determine the contribution of individual T cell population in the development of induced resistance, we have analyzed the in vitro proliferative capacity of phenotypically distinct T cell populations isolated from S1-immunized rats. We found that both CD8+ and CD8-CD4- T cells show striking proliferative responses when stimulated with S1 cells, whereas CD4+ T cells show only minimal responses. In addition, a significant proportion of the CD8-CD4- cells, after stimulated by S1 cells, became CD8+ and had a strong cytolytic activity toward S1 cells. These results suggest a contribution of double-negative splenic T cells in the regulatory circuit associated with autoimmune encephalomyelitis.
Animals↗