Amelioration of experimental autoimmune encephalomyelitis with anti-OX40 ligand monoclonal antibody: a critical role for OX40 ligand in migration, but not development, of pathogenic T cells

OX40 (CD134) and its ligand (OX40L) have been implicated in T cell activation and migration. In this study, we examined the contribution of these molecules to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) by administering a neutralizing mAb against murine OX40L (RM134L) to proteolipid protein (139-151) peptide-induced EAE in SJL mice. Administration of RM134L effectively ameliorated the disease in both actively induced and adoptively transferred EAE models. Histological examination showed that the RM134L treatment greatly reduced mononuclear cell infiltration into the spinal cord. The RM134L treatment did not inhibit the development of pathogenic T cells, given that proliferative response and IFN-gamma production by draining lymph node cells were not reduced or rather enhanced upon restimulation with proteolipid protein (139-151) in vitro, and these cells effectively transferred EAE to naive SJL mice. Flow cytometric analyses showed that the RM134L treatment inhibited the accumulation of OX40-expressing CD4(+) T cells and the migration of adoptively transferred CD4(+) T cells in the spinal cord. Immunohistochemical staining showed that OX40L was most prominently expressed on endothelial cells in the inflamed spinal cord. These results suggest that the OX40/OX40L interaction plays a critical role for the migration of pathogenic T cells into the CNS in the pathogenesis of EAE.     

T-cell activation induced by anti-CD3 × anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand

 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997     

Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.     

Amelioration of experimental lung metastasis in mice by therapy with anti-CD3 monoclonal antibodies

Summary Binding of CD3-specific antibodies to the TcR-CD3 complex results in T cell activation without the need for occupation of the T cell receptor (TcR) by its ligand. Murine T cells activated in this manner will kill a broad range of tumor targets but not normal lymphoblasts. We report here that non-specific cytolytic activity can be induced in vivo by a single i.p. injection of nonlytic 145-2C11 anti-CD3 monoclonal antibody. At least three populations of effector cells are activated in these mice. These are non-MHC(major histocompatibility complex) restricted cytotoxic T lymphocytes, activated natural killer cells, and lymphokine-activated killer cells. Anti-CD3 treatment is effective in significantly reducing the number of lung tumor nodules which form in mice inoculated with oncogenic ras-transfected syngeneic 10T1/2 fibroblasts. Anti-CD3-activated killer cells may, therefore, find a future role in cancer immunotherapy.     

Characterization of rat OX40 ligand by monoclonal antibody

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.     

A peptide mimetic of an anti-CD4 monoclonal antibody by rational design

The development of rational methods to design 'continuous' sequence mimetics of discontinuous regions of protein sequence has, to now, been only marginally successful. This has been largely due to the difficulty of constraining the recognition elements of a mimetic structure to the relative conformational and spatial orientations present in the parent molecule. Using peptide mapping to determine 'active' antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, we have developed a peptide mimic of an anti-CD4 antibody, containing antigen contact residues from multiple CDRs. The design described is a 27-residue peptide formed by juxtaposition of residues from 5 CDR regions. It displays an affinity for the antigen (CD4) of 0.9nM, compared to 2nM for the parent antibody ST40. Nevertheless, the mimetic shows low biological activity in an anti-retroviral assay.     

T cell immunity to lymphoma following treatment with anti-CD40 monoclonal antibody

In this study we demonstrate that treatment with anti-CD40 mAb eradicates a range of mouse lymphomas (BCL(1), A31, A20, and EL4), but only when used against i.v. tumor doses in excess of 10(7) cells. Only partial protection was seen against smaller tumor loads. We saw no evidence that anti-CD40 mAb changed the phenotype of the lymphomas or inhibited their growth in the initial period following treatment, but it did result in a rapid expansion of cytotoxic CD8(+) cells that was able to clear the neoplastic disease and provide long-term protection against tumor rechallenge. The CTL responses were blocked by mAb against a range of coreceptors and cytokines, including CD8, B7-1, B7-2, LFA-1, and IFN-gamma, but not CD4 or CTLA-4, indicating the presence of a conventional cellular Th1 response. Furthermore, we found evidence of cross-recognition between lymphomas (BCL(1) and A20) as measured by cytotoxicity and IFN-gamma responses in vitro and using tumor rechallenge experiments, suggesting common target Ags. Finally, although anti-CD40 was shown to stimulate NK cell killing, we could find no role for these cells in controlling tumor growth. These data underline the ability of anti-CD40 mAb to potentiate CTL responses and the potency of cellular immunity in eradicating large quantities of syngeneic tumor.     

Prevention of experimental autoimmune encephalomyelitis in common marmosets using an anti-IL-12p40 monoclonal antibody

The experimental autoimmune encephalomyelitis (EAE) model in the common marmoset approximates recognized features of the human disease multiple sclerosis (MS) with regard to its clinical presentation as well as neuropathological and radiological aspects of the lesions in brain and spinal cord. IL-12 is a proinflammatory cytokine that is produced by APC and promotes differentiation of Th1 effector cells. IL-12 is produced in the developing lesions of patients with MS as well as in EAE-affected animals. Previously it was shown that interference in IL-12 pathways effectively prevents EAE in rodents. In this study we report that in vivo neutralization of IL-12p40 using a novel Ab has beneficial effects in the myelin-induced EAE model in common marmosets. The Ab was injected i.v. at 7-day intervals starting well after immunization (day 14) and was continued until the end of the study (day 86). Stable levels of the Ab were measured 3 days after each injection throughout the study period. During this period anti-Ab responses could not be detected. We demonstrate that anti-IL-12p40 treatment has a protective effect on the neurological dysfunction as well as on neuropathological changes normally observed in the brain and spinal cord of EAE-affected individuals.     

Treatment with nonmitogenic anti-CD3 monoclonal antibody induces CD4+ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis

In vivo administration of anti-CD3 Ab induces both immune tolerance and undesirable side-effects resulting from nonspecific proinflammatory cytokine production. In the current study, we investigated the therapeutic potential of two structurally altered forms of the anti-CD3 Ab in ameliorating established experimental autoimmune encephalomyelitis. Administration of either a chimeric (NM-IgG3) or digestion product (NM-F(ab')2) form of the anti-CD3 Ab during established experimental autoimmune encephalomyelitis conferred significant protection from clinical disease progression and was associated with decreased Ag-specific T cell proliferation, cytokine production, and CNS inflammation. Interestingly, while this protection correlated with an increase in the frequency of CD4(+)CD25(+) regulatory T cells, neither prior depletion of regulatory T cells nor anti-TGF-beta treatment abrogated the treatment's efficacy. Importantly, both treatments induced normal levels of intracellular Ca(2+)-flux, but significantly diminished levels of TCR signaling. Consequent to this decreased level of TCR-mediated signaling were alterations in the level of apoptosis and CD4+ T cell trafficking resulting in a profound lymphopenia. Collectively, these results indicate that nonmitogenic anti-CD3 directly induces a state of immune unresponsiveness in primed pathogenic autoreactive effector cells via mechanisms that may involve the induction of T cell tolerance, apoptosis, and/or alterations in cell trafficking.     

Identification of a potent new pathogenic site in human retinal S-antigen which induces experimental autoimmune uveoretinitis in LEW rats

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of the eye which can be induced in LEW rats by immunization with either human or bovine S-antigen (S-Ag). In previous reports, two nonimmunodominant pathogenic sites were found using synthetic peptides corresponding to conserved sequences at amino acid residues 303-314 and 286-297 of the bovine sequence. In this report, a 20-residue synthetic peptide encompassing amino acids 343-362 located near the C-terminus was found to be highly immunopathogenic in LEW rats. The onset of EAU was observed at as early as 8 days when high doses of a peptide-encompassing residues 343-362 were used. EAU was elicited with as little as 0.5 microgram of peptide per animal. Smaller peptides from this region were also tested for uveitogenicity, further refining the site to 13 amino acids. Uveitogenic T cell lines were made to this site in two ways; first, by the in vitro selection of a bulk T cell line raised to human S-Ag with peptide 343-362. Second, by the in vitro selection of a peptide-specific line from an animal immunized with peptide 352-364, which corresponds to the minimal uveitogenic site. Both of these lines adoptively transferred EAU to LEW rats, further establishing the pathogenicity of this site. A proliferative site distinct from, but overlapping, the uveitogenic site was also found. The potent uveitopathogenicity of peptides from this region indicates that it is a major pathogenic site responsible for EAU induced in LEW rats by immunization with human S-Ag.     

Peritoneal exudate cells treated with calcitonin gene-related peptide suppress murine experimental autoimmune uveoretinitis via IL-10

Immunization with retinal Ag induces experimental autoimmune uveoretinitis (EAU) in mice. We investigated the suppression of murine EAU by peritoneal exudate cells (PEC) cultured with calcitonin gene-related peptide (CGRP). PEC derived from mice were treated with CGRP and residues 1-20 of human interphotoreceptor retinoid-binding protein (hIRBP 1-20). The hIRBP 1-20-immunized mice were injected i.v. with PEC treated with CGRP and hIRBP 1-20. After immunization, Ag-specific delayed hypersensitivity (DH) was measured and EAU was assessed histopathologically. Both EAU- and Ag-specific DH were suppressed by injection of PEC treated with CGRP (100 ng/ml) and hIRBP 1-20. However, hIRBP 1-20-mediated EAU was not suppressed by injection of PEC treated with CGRP and BSA. Both EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1-20 into splenectomized mice. In mice adoptively transferred spleen cells from hIRBP 1-20-immunized mice, EAU was also suppressed by injection of CGRP-treated PEC. EAU was markedly inhibited in hIRBP 1-20-immunized mice adoptively transferred T cells obtained from mice injected with hIRBP 1-20-pulsed, CGRP-treated PEC. Furthermore, EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1-20 when the recipient mice were given anti-IL-10 Ab i.p., or when the PEC were derived from IL-10 knockout mice. The present results indicate that PEC treated with CGRP suppress murine EAU in an Ag-specific manner, even in the efferent phase, and IL-10 secreted from PEC might play an important role in the CGRP-mediated suppression of murine EAU.     

FcR interactions do not play a major role in inhibition of experimental autoimmune encephalomyelitis by anti-CD154 monoclonal antibodies

It has been demonstrated that anti-CD154 mAb treatment effectively inhibits the development of experimental autoimmune encephalomyelitis (EAE). However, although it appears to prevent the induction of Th1 cells and reactivation of encephalitogenic T cells within the CNS, little information is available regarding the involvement of alternative mechanisms, nor has the contribution of Fc effector mechanisms in this context been addressed. By contrast, efficacy of anti-CD154 mAbs in models of allotransplantation has been reported to involve long-term unresponsiveness, potentially via activation of T regulatory cells, and recently was reported to depend on Fc-dependent functions, such as activated T cell depletion through FcgammaR or complement. In this study we demonstrate that anti-CD154 mAb treatment inhibits EAE development in SJL mice without apparent long-term unresponsiveness or active suppression of disease. To address whether the mechanism of inhibition of EAE by anti-CD154 mAb depends on its Fc effector interactions, we compared an anti-CD154 mAb with its aglycosyl counterpart with severely impaired FcgammaR binding and reduced complement binding activity with regard to their ability to inhibit clinical signs of EAE and report that both forms of the Ab are similarly protective. This observation was largely confirmed by the extent of leukocyte infiltration of the CNS; however, mice treated with the aglycosyl form may display slightly more proteolipid protein 139-151-specific immune reactivity. It is concluded that FcR interactions do not play a major role in the protective effect of anti-CD154 mAb in the context of EAE, though they may contribute to the full abrogation of peripheral peptide-specific lymphocyte responses.     

De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand

De novo sequencing of a full-length monoclonal antibody raised against OX40 ligand is described. Using a combination of overlapping complementary proteolytic and chemical digestions, with analysis by mass spectrometry and Edman degradation, both the heavy and light chains were fully sequenced. Particular attention was paid to those modifications that could be susceptible to degradation in the complementarity determining region and Fc region. An overview of the protocol is described, and suggestions for improvements to aid in such sequencing projects in the future are discussed.     

Inhibition of S-antigen induced experimental autoimmune uveoretinitis by oral induction of tolerance with S-antigen

The ability to prevent the expression of retinal SAg induced experimental autoimmune uveitis (EAU) in Lewis rats by oral administration of the SAg and SAg fragments was investigated. Oral administration of the SAg molecule prevented or markedly diminished the clinical appearance of SAg-induced disease as measured by ocular inflammation. Furthermore, oral administration of the SAg also markedly diminished uveitis induced by the uveitogenic M and N fragments of the SAg. M and N fragments were not effective in preventing SAg-induced EAU, although feeding the M fragment prevented disease induced by the M fragment. Oral administration of the SAg did not prevent myelin basic protein induced experimental autoimmune encephalomyelitis, whereas feeding myelin basic protein did. In vitro studies demonstrated a significant decrease in proliferative responses to the SAg in lymph node cells draining the site of immunization from fed vs nonfed animals. Furthermore, the addition of splenocytes from SAg-fed animals to cultures of a CD4+ SAg-specific cell line profoundly suppressed the cell line's response to the SAg, whereas these splenocytes had no effect on a purified protein derivative-specific cell line. The Ag-specific in vitro suppression was blocked by anti-CD8 antibody (OX-8) demonstrating that this suppression is dependent on CD8+ T-cells. These experiments demonstrate that Ag-specific immunomanipulation can be achieved in the EAU model by oral administration of the SAg and raise the possibility that such an approach may have practical clinical implications in uveitis as well as other human autoimmune diseases.     

IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.     

Suppression of development of experimental autoimmune myasthenia gravis with isogeneic monoclonal anti-idiotopic antibody

We have made use of isogeneic anti-idiotopic (anti-Id) monoclonal antibodies (mAb to modify experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. High-avidity anti-Id mAb HC-4A (Kd = 0.1 nM) and HC-29 (Kd = 0.1 nM) were produced against an anti-acetylcholine receptor (anti-AChR) Lewis-rat mAb 132A (Kd = 0.34 nM) that is capable of inducing passive-transfer EAMG. mAb HC-4A and HC-29 define separate framework Id cross-reactive with anti-AChR mAb recognizing different AChR epitopes. Animals were preinjected i.p. with either anti-Id mAb or with control mAb and then were actively immunized 2 wk later with purified AChR. All animals had elevated total serum anti-AChR antibody titers, despite the absence of weakness or decremental electromyographic findings. Animals preinjected with control mAb developed serum anti-AChR titers of 1.34 +/- 0.29 microM (mean +/- SEM) and reduced muscle AChR content to 30 percent of normal. Animals injected with 0.5 mg/kg of either anti-Id had significantly lower serum anti-AChR titers, 0.55 +/- 0.1, p less than 0.05, and normal muscle AChR content. Both the 132A Id and the anti-Id complementary to 132A were detected in the serum of all of the animals preinjected with this dose of either anti-Id HC-29 or HC-4A, whereas both were detected in a much smaller percentage of the animals receiving control mAb. These results show that pretreatment with anti-Id not only perturbs this Id-anti-Id network, but also suppresses the overall polyclonal anti-AChR response with resultant protection of actively immunized animals from EAMG.     

S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis

Two S-antigen-specific rat T cell lines expressing the T helper cell surface phenotype (W 3/25+, OX 8-) have been isolated from the spleen and lymph node cells of retinal S-antigen-immunized Lewis rats, one of which displayed neither clinical nor histopathologic signs of experimental autoimmune uveoretinitis. The other rat had recovered from severe experimental autoimmune uveoretinitis for 2 mo before isolation of the cell line. Both lines are specific for S-antigen presented by histocompatible antigen-presenting cells, and also respond in vitro to several of the peptides produced by cyanogen bromide cleavage of bovine retinal S-antigen. The lesions induced by the i.v. transfer of from 1 to 10 X 10(6) viable line cells involve the retina and pineal gland, as is found when Lewis rats are immunized with immunopathogenic doses of S-antigen. Histologic examination of the eyes and pineal glands revealed pathologic lesions typical of experimental autoimmune uveoretinitis, and consisted of marked infiltration of the retina and surrounding tissues and the pineal gland by lymphocytes and inflammatory cells. T cells capable of mediating autoimmune disease are clearly present and readily isolated from both asymptomatic and convalescent animals. No significant differences in specificity for the cyanogen bromide peptides of S-antigen or cell surface phenotype were found in the T cell lines isolated from these two rats, nor was any difference found in the specificity or titer of serum antibodies taken from the original rats for the cyanogen bromide peptides of S-antigen.     

Prevention and reversal of experimental autoimmune myasthenia gravis by a monoclonal antibody against acetylcholine receptor-specific T cells

We have recently described an algorithm to design, among others, peptides with complementarity contour to autoimmune epitopes. Immunization with one such peptide resulted in a monoclonal antibody (mAb), termed CTCR8, that specifically recognized Vbeta15 containing TCR on acetylcholine receptor (AChR) alpha-chain residue 100-116-specific T cells. CTCR8 was found to label the cell surface of AChR100-116-specific T cell lines and clones, immunoprecipitate the TCR from such cells, and block their proliferative responses to AChRalpha100-116. In the present report, we have found that there is a marked reduction in IFN-gamma and no effect on IL-10 production in a CTCR8-treated AChRalpha100-116-specific T cell line. Interestingly, when AChR100-116-primed, primary T cells were stimulated with peptide and treated with CTCR8, there was once again inhibition of IFN-gamma but also marked stimulation of IL-10 production. The change in the Th1/Th2 cytokine profile was paralleled by a reduction in AChR-specific IgG2a and IgM with no effect on IgG1. Remarkably, the most profoundly inhibited Ab population was that which causes experimental autoimmune myasthenia gravis (EAMG) by reaction with the main immunogenic region (alpha61-76) of the AChR. Based on these results, CTCR8 was tested for prophylactic and therapeutic effects in EAMG. EAMG induced by immunization with purified native Torpedo AChR was both inhibited and reversed by CTCR8. These findings suggest a means to produce therapeutic mAb for the treatment of autoimmune diseases.