Inhibition of rat autoimmune T cell activation by monoclonal antibodies

The essential requirement for adoptive transfer of autoimmune diseases such as experimental allergic encephalomyelitis (EAE) by T lymphoblasts from established T cell lines, is a prior activation of these cells by autoantigen or mitogen. We have investigated the possibility of modulating this activation process by using monoclonal antibodies directed against rat leukocyte differentiation antigens. We report here that antigen-driven activation of autoimmune, encephalitogenic T cells from established myelin basic protein (MBP)-specific rat T cell lines can be inhibited by some, but not all, antibodies against RT1.B Class II restriction elements. In addition, monoclonal antibodies with specificity for rat leukocyte common antigen (OX-1) and T cell differentiation antigens W3/13 and W3/25 are inhibitory, while monoclonal antibody OX-8 with specificity for T cytotoxic/suppressor cells has no effect. We also observed that concanavalin A-induced activation of the T cells is more resistant to the inhibitory effect of monoclonal antibodies, and can be blocked effectively only by antibody OX-1. This demonstration that autoimmune T cell function can be inhibited by monoclonal antibodies points the way in suggesting cellular targets for immunotherapeutic purposes.     

Coculture with autologous myotubes of cytotoxic T cells isolated from muscle in inflammatory myopathies.

T-cell lines were expanded from muscle of 10 patients with polymyositis, 5 with inclusion body myositis, 5 with dermatomyositis, and 5 with other muscle diseases. All cell lines uniformly expressed T-cell antigens, but not natural killer cell or B-cell antigens. The proportion of helper (CD4+) and cytotoxic (CD8+) T cells in the expanded lines was variable and showed no correlation with the diagnosis. Sixteen cell lines (6 polymyositis, 4 inclusion body myositis, 5 dermatomyositis, 1 other muscle disease) consisted predominantly of CD8+ T cells. None of these lines displayed natural killer-like cytotoxicity but all were capable of lectin-dependent cytotoxicity. Three of 6 polymyositis, 1 of 4 inclusion body myositis, and 1 of 5 dermatomyositis lines showed low but statistically significant cytotoxicity against autologous myotubes (6 to 27% specific 51Cr release; effector-target ratio, 20:1). The results demonstrate that functionally competent cytotoxic T cells can be expanded from muscle affected by inflammatory myopathies and are consistent with the hypothesis that some cytotoxic T cells recognize an autoantigen on myotubes. Further studies of this experimental system may define the molecular mechanism of T cell-mediated muscle fiber injury and may help to identify the relevant antigens.     

Neurological autoimmune disease and the trimolecular complex of T-lymphocytes

T-lymphocytes recognize antigen in a trimolecular complex: The T-cell receptor binds to a processed fragment of antigen that itself is bound to a major histocompatibility complex (MHC) molecule on the surface of an antigen-presenting cell. The trimolecular complex controls antigen-specific T-cell activation in normal and abnormal immune reactions. Recent progress in myasthenia gravis (MG) and experimental autoimmune encephalomyelitis (EAE) exemplifies this, leading to the following conclusions: (1) Autoimmune T cells may act by interfering with immunoregulation (as in MG) or by directly mediating autoimmune damage (as in EAE), or both. (2) In both diseases, the autoimmune T cells are clonally heterogeneous but recognize only a limited number of epitopes on the autoantigen (acetylcholine receptor in MG; myelin basic protein in EAE). Many of these epitopes can be defined as short peptide fragments of antigen, bound to a particular type of MHC molecule. (3) The MHC determines which peptides are recognized by autoimmune T cells in a given patient or inbred animal strain. (4) The discovery of the limited repertoire of autoimmune T cells has allowed considerable progress in the immunotherapy of EAE, using either monoclonal antibodies or cytotoxic T cells directed against clonotypic determinants on the autoaggressive T cells. (5) One obstacle to this approach in human disease is the polymorphism of the MHC in the species and the commensurate heterogeneity of autoimmune T cells.     

T cell response to myelin basic protein before and after treatment with interferon beta in multiple sclerosis.

Studies on the in vivo effects of interferon-beta (IFNbeta) therapy on autoreactive T cells have never been carried out in multiple sclerosis (MS). We investigated the T cell response to myelin basic protein (MBP), before and after IFN-beta therapy, raising MBP-specific T cell lines (TCL) from the peripheral blood of six MS patients with a satisfactory response to the treatment. IFNbeta did not affect the relative frequency and epitope specificity of the TCL. After IFNbeta therapy, the production of interleukin-4 was decreased in MBP-stimulated TCL while the secretion of interferon-gamma was increased in unstimulated TCL. Interleukin-10 and tumor necrosis factor-alpha did not show significant variations. This finding supports recent suggestions about the complexity of the T helper 1/T helper 2 paradigm in MS and other organ-specific autoimmune diseases. In fact, the beneficial effects of IFNbeta do not exclude an immunostimulatory action that may involve potentially autoreactive T cells. This has implications for future treatment options, including combination therapies.     

Epitopes common to trypanosomes (T. cruzi, T. dionisii and T. vespertilionis (Schizotrypanum]: astrocytes and neurons

An autoimmune mechanism is commonly invoked to explain the occurrence of neuronal destruction in the chronic phase of Chagas' disease. Monoclonal antibodies raised against T. dionisii (DION) and T. vespertilionis (VESP), and cross-reactive with T. cruzi recognize antigens in cultured cerebellar cells from embryonic and postnatal mice, as revealed by indirect immunofluorescence. Astrocytes (labelled with rabbit anti-GFAP antibody) showed positive reactions with DION 12.7, VESP 8.2 and VESP 9.3 while neurons (labelled with either tetanus toxin or anti-neuron-specific enolase antibody) reacted with the monoclonal antibody DION 10.1b. VESP 6.2 reacted with living cells of a subpopulation of neuronal or unidentifiable cell type. These cross-reactions may explain why not only neurons but also astrocytes may be involved in the autoimmune damage.     

Vaccination with autoreactive T cell clones in multiple sclerosis: overview of immunological and clinical data

Although the etiology and pathogenesis of Multiple Sclerosis (MS) remain elusive, accumulating evidence indicates that MS is a chronic inflammatory disease with an autoimmune component, mediated by autore-active T lymphocytes specific for myelin antigens. The triggering T cell autoantigen has not been identified yet, but recent immunological studies in MS and parallel experiments in experimental allergic encephalomyelitis (EAE), the animal model of MS, have indicated that myelin basic protein (MBP) can be considered as one of the major candidate autoantigens in the pathogenesis of the disease. Based on these observations, several therapeutic strategies have been developed aimed at the specific elimination or inactivation of MBP reactive T cells in MS. One of these approaches involves the immunization of MS patients with autologous attenuated autoreactive T cells to induce an immune response specifically targeted at these autoreactive T cells. This method, termed T cell vaccination, has been shown to prevent and treat EAE. We have recently conducted a pilot trial of T cell vaccination in a limited group of MS patients to evaluate the immunological responses to the injected cells. The data obtained indicate that this type of vaccination induces an effective anti-clonotypic T cell response leading to a specific depletion of circulating MBP reactive T cells. Preliminary data on the clinical effects are promising, encouraging further clinical trials. © 1996 Wiley-Liss, Inc.     

Targeting antigen-specific T cells by genetically engineered antigen presenting cells. A strategy for specific immunotherapy of autoimmune disease

We describe a strategy for specific immunotherapy of autoimmune disease based on targeting the antigen-specific T cells in an experimental model of myasthenia gravis. To address the problem of heterogeneity of the T cell repertoire, we have genetically engineered antigen presenting cells (APCs) to process and present epitopes of the autoantigen, acetylcholine receptor (AChR), to the entire spectrum of AChR-specific syngeneic T cells. APCs derived from BALB/c mice were stably transfected with cDNA for the key immunogenic domain of the AChR alpha-subunit, flanked by sequences of the lysosome-associated membrane protein (LAMP) that direct APCs to process and present the antigen via the MHC Class II pathway. Transfected APCs strongly stimulated AChR-specific T cells from BALB/c mice. Fas ligand, or antibody to Fas, abrogated the T cell response, by inducing apoptosis of the APC-stimulated T cells. The new results of this investigation are (1) that autoreactive T cells can be effectively targeted by autologous APCs that are engineered to present the relevant autoantigen, and (2) that these specifically targeted and activated T cells can be profoundly inhibited by agents that trigger the Fas-mediated apoptosis pathway. The present findings suggest that engineering APCs for simultaneous presentation of the autoantigen and delivery of FasL will provide a powerful strategy for the elimination of autoreactive T cells.     

Autoimmune T cell repertoire in optic neuritis and multiple sclerosis: T cells recognising multiple myelin proteins are accumulated in cerebrospinal fluid.

Monosymptomatic unilateral optic neuritis is a common first manifestation of multiple sclerosis. Abnormal T cell responses to myelin components including myelin basic protein (MBP), proteolipid protein (PLP), and myelin-associated glycoprotein (MAG) have been implicated in the pathogenesis of multiple sclerosis. Antigen-reactive T helper type 1 (Th1)-like cells that responded by interferon gamma (IFN-gamma) secretion on antigen stimulation in vitro were counted. Untreated patients with optic neuritis and multiple sclerosis had similarly raised levels of T cells recognising MBP, PLP, and MAG in peripheral blood. Such T cells were strongly enriched in CSF. None of these myelin antigens functioned as immunodominant T cell antigen characteristic for optic neuritis or multiple sclerosis. The autoimmune T cell repertoire was not more restricted in optic neuritis (as an example of early multiple sclerosis). The autoreactive T cell repertoires differed in blood compared with CSF in individual patients with optic neuritis and multiple sclerosis. No relations were found between specificity or quantity of autoreactive T cells in blood or CSF, and clinical variables of optic neuritis or multiple sclerosis, or occurrence of oligoclonal IgG bands in CSF. The role of raised MBP, PLP, and MAG reactive Th1-like cells found in optic neuritis and multiple sclerosis remains unexplained.     

Neonatal induction of myelin-specific Th1/Th17 immunity does not result in experimental autoimmune encephalomyelitis and can protect against the disease in adulthood

The neonatal immune system is believed to be biased towards T helper type 2 (Th2) immunity, but under certain conditions neonates can also develop Th1 immune responses. Neonatal Th2 immunity to myelin antigens is not pathogenic and can prevent induction of experimental autoimmune encephalomyelitis (EAE) in adulthood, but the consequences of neonatally induced Th1 immunity to self-antigens have remained unresolved. Here, we show that neonatal injection of mice with myelin antigens emulsified in complete Freund's adjuvant (CFA) induced vigorous production of IFN-gamma and IL-17, but not IL-5, consistent with myelin-specific Th1/Th17 immunity. Importantly, the myelin-specific Th1/Th17 cells persisted in the mice until adulthood without causing symptoms of EAE. Intraperitoneal, but not subcutaneous injection of neonates with myelin antigens protected against induction of EAE as adults. Intraperitoneally injected neonates showed a substantial decrease of the number and avidity of myelin-reactive Th17 cells, suggesting a decrease in IL-17 producing precursor cells as the mechanism of protection from EAE upon re-injection with myelin antigens as adults. The results could provide a rationale for the presence of autoreactive T cells found in healthy human individuals without autoimmune disease.     

Autoreactive T lymphocytes in multiple sclerosis: pathogenic role and therapeutic targeting

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), leading to demyelination. Accumulating evidence suggests that MS is an autoimmune disease, mediated by autoreactive T cells with specificity for myelin antigens. The identity of the brain antigens, which are the primary targets of the autoimmune process remains unknown, but myelin basic protein (MBP) is a likely candidate. We will overview some of the experimental evidence, suggesting that MBP reactive T cells hold a central position in the pathogenesis of MS, and discuss how these autoreactive T cells can be therapeutically targeted by T cell vaccination.     

Lymphocyte vaccination against experimental autoimmune encephalomyelitis: evaluation of vaccination protocols

Repeated vaccination with encephalitogenic but not other T cell lines could effect marked resistance to 'active' experimental autoimmune encephalomyelitis (EAE) induced by injection of GP-BP in adjuvant. Partial resistance to active EAE was observed in rats recovered from 'passive' line-mediated EAE and in rats vaccinated with T cells attenuated by irradiation or ganglioside treatment. However, no resistance was observed in animals given low doses of activated encephalitogenic T cells. Treatment with hydrostatic pressure alone was found to be ineffective as a means of attenuation, and vaccination with pressure-treated encephalitogenic T cells actually induced mild signs of EAE. However, vaccination with cells that were first pressure treated and then irradiated prevented both clinical and histologic signs of active EAE. In contrast, protection against passive EAE appeared to be clonotypic. Lymphocyte vaccination induced delayed type hypersensitivity (DTH) reactions against autologous T cells, mostly to shared antigens, demonstrating the immunogenicity of multiple antigens on the vaccinating cells.     

Immunopathogenesis and treatment of the guillain-barré syndrome—part I

The etiology of the Guillain-Barré syndrome (GBS) still remains elusive. Recent years have witnessed important advances in the delineation of the mechanisms that may operate to produce nerve damage. Evidence gathered from cell biology, immunology, and immunopathology studies in patients with GBS and animals with experimental autoimmune neuritis (EAN) indicate that GBS results from aberrant immune responses against components of peripheral nerve. Autoreactive T lymphocytes specific for the myelin antigens PO and P2 and circulating antibodies to these antigens and various glycoproteins and glycolipids have been indentified but their pathogenic role remains unclear. The multiplicity of these factors and the involvement of several antigen nonspecific proinflammatory mechanisms suggest that a complex interaction of immune pathways results in nerve damage. Data on disturbed humoral immunity with particular emphasis on glycolipid antibodies and on activation of autoreactive T lymphocytes and macrophages will be reviewed. Possible mechanisms underlying initiation of peripheral nerve-directed immune responses will be discussed with particular emphasis on the recently highlighted association with Campylobacter jejuni infection.© 1995 John Wiley &Sons, Inc.     

Surface-binding autoantibodies to cerebellar neurons in opsoclonus syndrome

Childhood opsoclonus-myoclonus syndrome can occur with or without associated neuroblastoma. An autoimmune pathogenesis has been discussed for both forms. We show here that the majority of children with opsoclonus-myoclonus syndrome (10/14) have autoantibodies binding to the surface of isolated rat cerebellar granular neurons. In some patients, these antibodies are masked by IgG binding to ubiquitous surface antigens, which could be removed by preincubation with the nonneuronal control cell line HEK 293. A newly introduced competitive binding assay showed that the surface binding is directed against the same autoantigen in different patients. Therefore, we hypothesize that opsoclonus-myoclonus syndrome may be the result of an autoimmune process against a neuronal surface protein.     

Intra-CNS activation by antigen-specific T lymphocytes in experimental autoimmune encephalomyelitis

Identification and quantitation of autoreactive T lymphocytes is crucial in order to understand the pathogenesis of autoimmune diseases. We used flow cytometry to analyze autoantigen-specific T cellular responses in the well characterized rat experimental autoimmune encephalomyelitis (EAE) model. Cells isolated from both the central nervous system (CNS) tissue and peripheral lymph nodes were analyzed directly ex vivo or after short term in vitro culture with specific autoantigen. CNS infiltrating T lymphocytes displaying an interferon-gamma response to selected encephalitogenic myelin protein epitopes were measured kinetically during an individual disease episode and also between relapses in a chronic rat EAE model. One of the EAE models used displays a restriction towards TCRBV8S2 chain usage by the encephalitogenic T cells. In this model, in vitro production of intracellular interferon-gamma was selectively detected within this T cell subset derived from both the CNS and peripheral lymph nodes. Furthermore, antigen-specific cells infiltrating the CNS in this model produced several-fold higher amounts of interferon-gamma upon antigen stimulation and displayed a significantly increased in vivo proliferation compared with peripheral lymphocytes. These data thus directly demonstrates that T cells stimulated by a specific autoantigen in the periphery primarily acquire effector functions in the cellular environment of the target organ of the autoantigen.     

Cerebrospinal fluid cells in multiple sclerosis and other neurological diseases: an immunocytochemical study

Summary Determinations of mononuclear cell subsets in cerebrospinal fluid (CSF), using monoclonal antibodies against surface antigens which identified pan T-cells, helper/inducer T-cells, cytotoxic/suppressor T-cells and Ia-positive cells, were performed in patients with multiple sclerosis (MS), other neuroimmunological diseases (NID), infectious diseases (INF) of the central nervous system and with other neurological diseases. Whereas there was an elevated helper T/suppressor T ratio in CSF of patients with NID (Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, cerebral vasculitis), no other significant differences could be detected between the different groups of patients. Our results suggest that analysis of these mononuclear cell subsets in CSF is not helpful in discriminating between MS and other neurological diseases and that in MS patients changes in disease activity are not clearly indicated by fluctuations in the different CSF cell subsets. Further studies will be needed to confirm our findings in NID patients and to understand the diagnostic and theoretical implications.     

On the role of astrocytes in polyclonal T cell activation

Murine astrocytes have been identified to possess accessory cell functions, which are thought to be regulated by the inducible expression of surface Ia molecules. The accessory cell functions include the induction of syngeneic and allogeneic T lymphocyte responses, the cytotoxic T cell activation towards haptens and the antigen-induced stimulation of helper T cell lines. In this report, astrocytes initially being Ia antigen-negative cells were found to restore the phytohaemagglutinin (PHA) response of mouse lymph node cells depleted of accessory cells by treatment with leucin methyl ester (Leu ME). Antisera against Ia determinants did not inhibit the PHA response or the release of interleukin-1 (IL-1) by activated astrocytes. Thus, the presence of Ia antigens is not required for mitogen-induced polyclonal T cell activation. In addition to IL-1 release, astrocytes may favour cell-cell contacts necessary for mitogen-induced polyclonal T cell response.     

Murine monoclonal anti-myelin basic protein (MBP) antibodies inhibit proliferation and cytotoxicity of MBP-specific human T cell clones

Myelin basic protein (MBP)-specific T cell clones, isolated from two patients with multiple sclerosis, expressed the CD4+ phenotype and induced MBP-dependent cytolysis of autologous Epstein-Barr virus (EBV)-transformed B cells. The proliferation and cytolytic activity of the T cell clones were inhibited by four of a panel of five murine monoclonal anti-MBP antibodies in a dose-dependent manner. An isotype-matched antibody with an irrelevant specificity did not have such an effect. These MBP-specific monoclonal antibodies did not block phytohemagglutinin-induced T cell proliferation or allospecific cytotoxicity. These results suggest that some antibodies directed at the autoantigen MBP may play a regulatory role in T cell activation, rather than a pathogenic role, for which there is currently little supporting evidence.     

The effect of cyclophosphamide on T lymphocytes and T lymphocyte subsets in patients with chronic progressive multiple sclerosis

ABSTRACT— The lymphocytes in peripheral blood and cerebrospinal fluid of patients with chronic progressive multiple sclerosis (MS) were characterized with monoclonal antibodies to surface antigens of T cells, helper/inducer T cells and suppressor/cytotoxic T cells. The influence of cyclophosphamide treatment on these immune parameters was investigated.
Compared to healthy persons, the mononuclear cell fraction of the peripheral blood of patients with chronic progressive MS consisted of normal %s of T cells and helper/inducer T cells, but decreased %s of suppressor/cytotoxic T lymphocytes. Intensive as well as chronic treatment of MS patients with cyclophosphamide resulted in a decline in the %s of T cells and helper/inducer T cells, whereas the %s of suppressor/cytotoxic T cells returned to normal. In cerebrospinal fluid, cyclophosphamide also induced a relative decrease in the % of helper/inducer T cells and an increase in the % of suppressor/cytotoxic T cells compared to untreated MS patients. Intensive as well as chronic therapy with cyclophosphamide both led to a significant decrease in the absolute number of T cells and T cell subsets in the blood of the patients.     

Decreased suppressor-inducer T lymphocytes in multiple sclerosis and other neurological diseases

Decreased numbers of CD4+CD45R+ suppressor-inducer T cells have been reported in patients with a variety of autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis but not in patients with other neurological diseases. We now report our findings using murine monoclonal antibodies and two-color flow cytometric analysis on CD4+CD45R+ T cells in 22 patients with chronic progressive multiple sclerosis, 23 patients with other neurological diseases and 42 normal, healthy controls. Suppressor-inducer T cells were significantly reduced (p less than 0.001) in both patients with multiple sclerosis and other neurological diseases as compared to controls. Both patient populations had elevated helper T cell subset ratios. Thus, our data suggests that decreases in suppressor-inducer T cells may represent a common immunological defect among autoimmune and presumably non-autoimmune neurological disorders.