Subramaniam Sriram

Experimental allergic encephalomyelitis: Neutralizing antibody to TGFβ1 enhances the clinical severity of the disease

Experimental allergic encephalomyelitis (EAE) is a well established model for the human autoimmune disease multiple sclerosis. Recently, we and others have shown that the administration of TGF beta is therapeutically effective in reducing incidence and severity of EAE. Here we show that the addition of anti-TGF beta 1 to myelin basic protein (MBP)-activated lymph node cells enhance the T cell proliferative response by 28% in vitro and in vivo and that injections of anti-TGF beta 1 antibody worsen EAE both in incidence and severity. Further, an inverse relationship was observed in the amount of IL-2 and TGF beta detected in MBP stimulated culture supernatants. We show that IL-2 decreases from 248 U/ml at 48 h to non-detectable at 96 h, while TGF beta increases from 0.5 ng/ml to 1.2 ng/ml, respectively. These observations further indicate a role for endogenous TGF beta 1 in the immunoregulation of EAE.

Transforming growth factor-β1 differentially regulates proliferation and MHC class-II antigen expression in forebrain and brainstem astrocyte primary cultures

To facilitate investigation of cytokine regulation of reactive astrogliosis, primary astrocyte cultures from neonatal murine forebrain and brainstem were established. Forebrain and brainstem astrocytes proliferated at a similar rate under basal culture conditions, and both were growth-inhibited by treatment with recombinant murine interferon-gamma. The growth of cultured brainstem astrocytes was significantly enhanced by exposure to recombinant human transforming growth factor-beta 1. In contrast, proliferation of forebrain astrocytes was not significantly affected by transforming growth factor-beta 1. The disparate responses of brainstem and forebrain astrocytes to transforming growth factor-beta 1 treatment were not limited to effects on cell growth, since transforming growth factor-beta 1 could block interferon-gamma-induced MHC class-II antigen expression on cultured brainstem astrocytes but not on forebrain cells. Results could not be attributed to use of an heterologous cytokine/cellular target system, since similar variability in transforming growth factor-beta 1 modulation of major histocompatibility complex antigen expression could be demonstrated using two human astrocytoma cell lines. This report is the first to document mitogenic response to transforming growth factor-beta 1 for neuroepithelial cells. The role of transforming growth factor-beta 1 in regulating aspects of reactive astrogliosis, particularly in the context of inflammatory demyelination, requires further investigation. Furthermore, these studies may provide insight into regional variability in the sequelae of inflammation within the central nervous system.

Treatment of chronic relapsing experimental allergic encephalomyelitis with the intravenous administration of splenocytes coupled to encephalitogenic peptide 91–103 of myelin basic protein☆

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) is an autoimmune demyelinating disease of the central nervous system and serves as an experimental model of human multiple sclerosis. Amino acid residues p91-103 of myelin basic protein are encephalitogenic in SJL mice and transfer of T cell lines that recognize this epitope results in CR-EAE. We show here that coculture of T cells in the presence of p91-103 that has been chemically cross-linked to the antigen presenting cells renders the T cell lines tolerant to the antigen. Injection of p91-103 coupled splenocytes into animals that had received encephalitogenic p91-103 reactive T cells significantly reduced the incidence and severity of EAE. Furthermore, treatment of mice with a single injection of antigen coupled splenocytes after they had recovered from their initial paralytic attack prevented the development of subsequent clinical relapses in all animals. These studies indicate that this effect is long lasting and can be successfully accomplished in an established autoimmune disease. Hence this form of immunotherapy may be considered as a therapeutic modality in the treatment of autoimmune diseases when the autoantigens are known.

Analysis of TCR Vβ gene usage and encephalitogenicity of myelin basic protein peptide p91–103 reactive T cell clones in SJL mice: Lack of evidence for V gene hypothesis☆

We have analyzed the epitope specificity and encephalitogenicity of peptides that span the C terminus of MBP, p84-103. Our studies show that multiple antigenic epitopes with disease-inducing capacity exist in SJL mice. Three peptides that span this region were examined and found to be immunogenic. However, the mode of immunization (active or passive) determined the incidence and severity of EAE. In our experiments adoptive transfer of p91-103-reactive T cell lines was most consistent in the development of disease. Interestingly, the response to peptides p89-101, p91-103, and p84-102 was absent following immunization with MBP. This suggests that although p91-103 and p89-101 were encephalitogenic they were not the major immunogenic epitopes following immunization with MBP. Analysis of a panel of eight p91-103-reactive T cell clones showed significant heterogeneity in the fine specificity, the TCR V beta gene usage, and in their ability of transfer EAE. These studies suggest that in SJL mice the epitopes involved in the pathogenesis of disease are multiple and there is no clear correlation between encephalitogenicity and TCR V beta gene usage. These observations argue against the presence of a dominant TCR V beta gene in the pathogenesis of EAE in SJL mice.

In vivo immunomodulation by monoclonal anti-L3T4: 1. Effects on humoral and cell-mediated immune response☆

The in vivo administration of monoclonal anti-L3T4 antibody has been shown to be an effective preventative and, in some cases, therapeutic treatment for several murine models of autoimmune disease. This report deals with the effect of such treatments on humoral and cell-mediated responses to T-dependent antigens. Both the primary and secondary IgG responses to tetanus toxoid were inhibited when anti-L3T4 was administered prior to immunization, but it was ineffective in modulating an ongoing IgG response. Cell-mediated immunity, as detected by in vitro antigen-specific proliferative responses, was inhibited only if anti-L3T4 was given prior to immunization. It was not effective if treatment was delayed until 48 hr prior to lymph node harvest even though greater than 90% of L3T4+ lymph node cells were depleted by this treatment. The refractory behavior of the lymph node cells to anti-L3T4 treatment was not exhibited by antigen-primed cells obtained from peripheral blood or spleen. The importance of these findings with regard to antibody therapy for chronic autoimmune disease is discussed.

In vivo depletion of Lyt-2 cells fails to alter acute and relapsing EAE

The cellular mechanisms of recovery and relapses in experimental allergic encephalomyelitis (EAE) are not known. In order to determine the role of the suppressor/cytotoxic T lymphocytes (Lyt-2+) in EAE we studied the effect of in vivo depletion of this subset using monoclonal antibodies. Intraperitoneal injection of 1 mg of monoclonal antibody 2.43 resulted in rapid depletion of Lyt-2+ cells from lymph node, spleen and blood. Depletion of this subset had no effect on the kinetics of development, severity, and duration of acute EAE. Furthermore, following recovery from acute EAE administration of 2.43 did not result in development of relapses that were different in onset or severity from control animals. These results suggest that T cells of the Lyt-2 phenotype do not play a significant role in the immunoregulation of EAE.

In Vivo Modulation of Murine Collagen Induced Arthritis

The effects of in vivo modulation of murine collagen induced arthritis with monoclonal anti-CD4 antibodies, monoclonal anti-Ia antibodies, and gamma interferon are reviewed. We detail the mechanism of action of monoclonal anti-CD4 antibody on humoral and cell mediated immune responses and discuss the implications for designing therapeutic strategies. To further explore the induction of collagen induced arthritis, a syngeneic cell transfer system using collagen primed T lymphocytes is described. This cell transfer system provides an opportunity to study the role of CD4 positive T lymphocytes in arthritis induction during a short, defined time period.

Spontaneous development in vitro of a myelin basic protein-specific suppressor T cell line

T cell lines to myelin basic protein (MBP) developed following in vitro culture cause experimental allergic encephalomyelitis (EAE) upon transfer into naive recipient mice. We have, however, repeatedly observed that MBP-specific T cell lines lose their ability to transfer EAE after 40 days in culture. Analyses of such cell lines failed to show any differences in their proliferative responses to antigen, or in the secretion of interleukin-2 (IL-2) and/or IL-4 when compared to their encephalitogenic counterparts. In contrast, examinations of T cell receptor (TCR) beta-chain gene rearrangement patterns showed sequential changes in the clonal population of cells concomitant with the loss of encephalitogenic function. Furthermore, transfer of a non-encephalitogenic, genotypically altered cell line after long-term in vitro culture into mice challenged with MBP suppressed the development of EAE. These findings suggest that the development of such putative regulatory cells in vivo may be involved in the recovery in EAE.

Clonal diversity of myelin basic protein-specific T lymphocytes

A panel of myelin basic protein (MBP)-specific, class II major histocompatibility complex (As)-restricted T-cell clones were established from SJL/J mice. Three clonotypes, based on their responses to guinea pig MBP and its peptide fragments, were observed. Clonotype I cells, represented by clones HS.6, HS.D2, HS.8, HS.E10, and HS.C1, were reactive to the encephalitogenic C-terminal fragment of MBP, amino acid residues 89–169. Clonotype II, represented by clone HS.E3, was reactive to fragments containing residues 43–88, and clones HS.D12 and HS.C7, representing clonotype III cells, responded to the whole molecule only. Three clones from clonotype I were capable of transferring both clinical and histological signs of experimental allergic encephalomyelitis (EAE) into naive mice. Southern blot analysis of T-cell receptor β-chain genes using Jβ1- and Jβ2-specific probes showed that the rearrangement pattern was unique in each of the clones. These results suggest that the development of EAE may represent an autoaggressive polyclonal T-cell response.

Haplotype-specific inhibition of homing of radiolabeled lymphocytes in experimental allergic encephalomyelitis following treatment with anti-IA antibodies.

In vivo treatment with anti-IA antibodies has been shown to induce a haplotype-specific inhibition of EAE when the disease was following passive transfer of MBP-sensitized T cells. In order to determine the mechanism by which anti-IA antibody prevents passively transferred EAE, the homing of radiolabeled cells to the brain following anti-IA therapy was studied. Administration of anti-IA antibodies at the earliest onset of clinical signs of EAE prevented the homing of radio-labeled cells to the brain. In F1 (Balb/c x SJL/J) mice that developed EAE and received anti-IAs antibody there was a decreased homing of radiolabeled cells when compared to animals that received anti-IAd antibody. In addition, there was preferential expression of IAs antigen, over IAd antigen on capillary endothelium of the brain. The differential expression of IA antigens and the homing of radiolabeled cells in F1 (SJL x Balb/c) mice could in part explain the haplotype-specific suppression of disease following treatment with anti-IA antibodies.