Characterization of a surrogate murine antibody to model anti-human CD3 therapies

Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.     

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.     

Gatekeeper role of brain antigen‐presenting CD11c+ cells in neuroinflammation

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen‐presenting cells, dendritic cells. Applying intravital two‐photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen‐presenting CD11c⁺ cells, which preferentially interact with Th17 cells. IL‐17 expression correlates with expression of GM‐CSF by T cells and with accumulation of CNS CD11c⁺ cells. These CD11c⁺ cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c⁺ cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c⁺ cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS.     

Extracellular αB-crystallin modulates the inflammatory responses

Neuroinflammation is considered a challenging clinical problem. Chronic inflammatory responses play important roles in the onset and progression of various neurodegenerative diseases, including multiple sclerosis (MS). Previous studies have shown that astrocytes express small heat shock protein αB-crystallin (CRYAB) which is capable of inhibiting inflammatory responses in astrocytes per se. However, the underlying mechanisms of CRYAB-induced modulation of neuroinflammation are still not fully understood. In the present study, we investigated the role of extracellular CRYAB in the interaction between microglia and astrocytes in the context of MS-associated neuroinflammation. We found that the expression of CRYAB was profoundly increased in EAE mice. CRYAB was preferentially expressed in astrocytes and could be secreted via exosomes. Levels of exosomal CRYAB secreted from astrocytes were markedly increased under stress conditions. Furthermore, incubation of immortalized astrocytes or microglia cell lines with CRYAB remarkably suppressed astrocytes and microglia-mediated inflammatory responses in both autocrine and paracrine manners. Our results reveal a novel function for extracellular CRYAB in the regulation of neuroinflammation. Targeting extracellular CRYAB-modulated neuroinflammation is a potential therapeutic intervention for MS.     

Potential roles and targeted therapy of the CXCLs/CXCR2 axis in cancer and inflammatory diseases

The chemokine receptor CXCR2 and its ligands are implicated in the progression of tumours and various inflammatory diseases. Activation of the CXCLs/CXCR2 axis activates multiple signalling pathways, including the PI3K, p38/ERK, and JAK pathways, and regulates cell survival and migration. The CXCLs/CXCR2 axis plays a vital role in the tumour microenvironment and in recruiting neutrophils to inflammatory sites. Extensive infiltration of neutrophils during chronic inflammation is one of the most important pathogenic factors in various inflammatory diseases. Chronic inflammation is considered to be closely correlated with initiation of cancer. In addition, immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) against T cells attenuate the anti-tumour effects of T cells and promote tumour invasion and metastasis. Over the last several decades, many therapeutic strategies targeting CXCR2 have shown promising results and entered clinical trials. In this review, we focus on the features and functions of the CXCLs/CXCR2 axis and highlight its role in cancer and inflammatory diseases. We also discuss its potential use in targeted therapies.     

Gene-Expression Profiling of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that serves as an experimental tool for studying the etiology, pathogenesis, as well as new therapeutic approaches of multiple sclerosis (MS). EAE is a polygenic chronic inflammatory demyelinating disease of the nervous system that involves the interaction between genetic and environmental factors. Previous studies have identified multiple quantitative trait loci (QTL) controlling different aspects of disease pathogenesis. However, progress in identifying new susceptibility genes outside the MHC locus has been slow. With the advent of new global methods for genetic analysis such as large-scale sequencing, gene expression profiling combined with classic linkage analysis and congenic and physical mapping progress is considerably accelerating. Here we review our preliminary work on the use of gene expression mapping to identify new putative genetic pathways contributing to the pathogenesis of EAE.     

The role of antigen presenting cells in multiple sclerosis

Multiple sclerosis (MS) is a debilitating T cell mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

Direct and indirect effects of microbiota-derived metabolites on neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are highly influenced by changes in the microbiota and of microbiota-derived metabolites, including short chain fatty acids, bile acids, and tryptophan derivatives. This review will discuss the effects of microbiota-derived metabolites on neuroinflammation driven by central nervous system-resident cells and peripheral immune cells, and their influence on outcomes of EAE and MS.     

The association between EAE development in mice and the production of autoantibodies and abzymes after immunization of mice with different antigens

We have previously shown that immunization of C57BL/6 mice, prone to spontaneous development of experimental autoimmune encephalomyelitis (EAE), with three antigens (MOG_35-55, DNA-histone complex or DNA-methylated BSA complex), alters the differentiation profiles of bone marrow haematopoietic stem cells (HSCs). These are associated with the production of autoantibodies (auto-Abs) against these antigens and the formation of abzymes hydrolysing DNA, MOG, myelin basic protein (MBP) and histones. Immunization of mice with antigens accelerates the development of EAE. This work is the first to analyse the ratio of auto-Abs without and with catalytic activities at different stages of EAE development (onset, acute and remission phases) after immunization of mice with the three specific antigens. Prior to immunization and during spontaneous in-time development of EAE, the concentration of auto-Abs against MBP, MOG, histones and DNA and activities of IgG antibodies in the hydrolysis of substrates increased in parallel; correlation coefficients = +0.69-0.94. After immunization with MOG, DNA-histone complex or DNA-met-BSA complex, both positive (from +0.13 to +0.98) and negative correlations (from −0.09 to −0.69) were found between these values. Our study is the first showing that depending on the antigen, the relative amount of harmful auto-Abs without and abzymes with low or high catalytic activities may be produced only at onset and in acute or remission phases of EAE. The antigen governs the EAE development rate, whereby the ratio of auto-Abs without catalytic activity and with enzymatic activities of harmful abzymes hydrolysing MBP, MOG, histones and DNA varies strongly between different disease phases.