Increasing recognition of the role of B cells in the adaptive immune response makes B cells an important therapeutic target in autoimmunity. Numerous current and developmental immunotherapies target B cells for elimination through recognition of cell-surface proteins expressed specifically on B cells, in particular CD19 and CD20. Similarities and differences in the function and expression of these two molecules predict some shared, and some distinct, pharmacological effects of agents targeting CD19 versus CD20, potentially leading to differences in the clinical safety and efficacy of such agents. Here, we review current knowledge of CD19 and CD20 function and biology, survey current and developmental therapies that target these molecules, and discuss potential differences in elimination of B cells by drugs that target CD19 versus CD20, with particular focus on the central nervous system autoimmune diseases multiple sclerosis and neuromyelitis optica. The principles and mechanisms herein discussed might also be relevant to a variety of other nervous system autoimmune disorders, including NMDA (N-methyl-D-aspartate) receptor encephalitis, transverse myelitis and myasthenia gravis.

While it was long held that T cells were the primary mediators of multiple sclerosis (MS) pathogenesis, the beneficial effects observed in response to treatment with Rituximab (RTX), a monoclonal antibody (mAb) targeting CD20, shed light on a key contributor to MS that had been previously underappreciated: B cells. This has been reaffirmed by results from clinical trials testing the efficacy of subsequently developed B cell-depleting mAbs targeting CD20 as well as studies revisiting the effects of previous disease-modifying therapies (DMTs) on B cell subsets thought to modulate disease severity. In this review, we summarize current knowledge regarding the complex roles of B cells in MS pathogenesis and current and potential future B cell-directed therapies.

Activation of inflammatory cells is central to the pathogenesis of autoimmune demyelinating diseases of the peripheral nervous system. The novel chimeric compound quinpramine--generated from imipramine and quinacrine--redistributes cholesterol rich membrane domains to intracellular compartments. We studied the immunological and clinical effects of quinpramine in myelin homogenate induced Lewis rat experimental autoimmune neuritis (EAN), a model system for acute human inflammatory neuropathies, such as the Guillain-Barré syndrome. EAN animals develop paresis of all limbs due to autoimmune inflammation of peripheral nerves. Quinpramine treatment ameliorated clinical disease severity of EAN and infiltration of macrophages into peripheral nerves. It reduced expression of MHC class II molecules on antigen presenting cells and antigen specific T cell proliferation both in vitro and in vivo. Quinpramine exerted its anti-proliferatory effect on antigen presenting cells, but not on responder T cells. Our data suggest that quinpramine represents a candidate pharmaceutical for inflammatory neuropathies.

Lymphocyte homing into the intestine is mediated by binding of leukocytes to mucosal addressin cell adhesion molecule 1 (MAdCAM-1), expressed on endothelial cells. Currently, the immune system of the gut is considered a major modulator not only of inflammatory bowel disease, but also of extra-intestinal autoimmune disorders, including multiple sclerosis (MS). Despite intense research in this field, the exact role of the intestine in the pathogenesis of (neuro-)inflammatory disease conditions remains to be clarified. This prompted us to investigate the role of MAdCAM-1 in immunological processes in the intestine during T cell-mediated autoimmunity of the central nervous system (CNS). Using the experimental autoimmune encephalomyelitis model of MS, we show that MAdCAM-1-deficient (MAdCAM-1-KO) mice are less susceptible to actively MOG35-55-induced disease. Protection from disease was accompanied by decreased numbers of immune cells in the lamina propria and Peyer's patches as well as reduced immune cell infiltration into the spinal cord. MOG35-55-recall responses were intact in other secondary lymphoid organs of MAdCAM-1-KO mice. The composition of specific bacterial groups within the microbiome did not differ between MAdCAM-1-KO mice and controls, while MAdCAM-1-deficiency severely impaired migration of MOG35-55-activated lymphocytes to the gut. Our data indicate a critical role of MAdCAM-1 in the development of CNS inflammation by regulating lymphocyte homing to the intestine, and may suggest a role for the intestinal tract in educating lymphocytes to become encephalitogenic.

The goal of this study was to investigate the role of CD 19+ B cells within the brain and spinal cord during CNS autoimmunity in a peptide-induced, primarily T-cell-mediated experimental autoimmune encephalomyelitis (EAE) model of MS. We hypothesized that CD19+ B cells outside the CNS drive inflammation in EAE.

Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c+ cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre+/-ITGA4fl/fl C57BL/6 mice to selectively delete α4-integrin in CD11c+ cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c+ cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre+/-ITGA4fl/fl mice. In active EAE, a delayed disease onset was observed in CD11c.Cre+/-ITGA4fl/fl mice, during which CD11c+CD88+ cells were sequestered in the blood. Upon clinical EAE onset, CD11c+CD88+ cells appeared in the CNS and expressed CD317+ In adoptive transfer experiments, CD11c.Cre+/-ITGA4fl/fl mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11c+CD88+CD317+ cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of ITGAX (CD11c), C5AR1 (CD88), and BST2 (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice.

Multiple sclerosis (MS) is a chronic inflammatory-demyelinating disease of the central nervous system of a putative autoimmune etiology. Although the exact pathogenic mechanisms underlying demyelination and axonal damage in MS are not fully understood, T-cells are believed to play a central role in the pathogenesis of the disease. Daclizumab is a humanized binding monoclonal antibody that binds to the Tac epitope on the α-subunit (CD25) of the interleukin-2 (IL-2) receptor, thus effectively blocking the formation of the high-affinity IL-2 receptor, which is expressed mainly on T-cells. A series of clinical trials in patients with relapsing MS demonstrated a profound effect of daclizumab on inflammatory disease activity and improved clinical outcomes compared with placebo or interferon-β, which led to the recent approval of daclizumab (Zinbryta™) for the treatment of relapsing forms of MS. Enhancement of endogenous mechanisms of immune regulation rather than inhibition of effector T-cells might explain the effects of daclizumab in MS. These include expansion and improved function of regulatory CD56bright NK cells, inhibition of the early activation of T-cells through blockade of IL-2 transpresentation by dendritic cells and reduction in the number of intrathecal proinflammatory lymphoid tissue inducer cells. The enhanced efficacy of daclizumab is accompanied by an increased frequency of adverse events and risks of serious adverse events, thus placing it as a second-line therapy and calling for the implementation of a strict risk management program. This review details the mechanisms of action of daclizumab, discusses its efficacy and safety in patients with MS, and provides an insight into the place of this novel therapy in the treatment of MS.

Natalizumab blocks α4-integrin-mediated leukocyte migration into the central nervous system (CNS). It diminishes disease activity in multiple sclerosis (MS), but carries a high risk of progressive multifocal encephalopathy (PML), an opportunistic infection with JV virus that may be prompted by diminished CNS immune surveillance. The initial host response to viral infections entails the synthesis of type I interferons (IFN) upon engagement of TLR3 receptors. We hypothesized that TLR3 agonism reestablishes CNS immune competence in the setting of α4-integrin deficiency.

Aquaporin 4 (AQP4) is considered a putative autoantigen in patients with Neuromyelitis optica (NMO), an autoinflammatory disorder of the central nervous system (CNS). HLA haplotype analyses of patients with NMO suggest a positive association with HLA-DRB1* 03:01. We previously showed that the human (h) AQP4 peptide 281-300 is the dominant immunogenic determinant of hAQP4 in the context of HLA-DRB1*03:01. This immunogenic peptide stimulates a strong Th1 and Th17 immune response. AQP4281-300-specific encephalitogenic CD4+ T cells should initiate CNS inflammation that results in a clinical phenotype in HLA-DRB1*03:01 transgenic mice.

To determine the capacity, effectiveness, efficiency, and reliability of select tissue dissociation methods to isolate mononuclear cells from the CNS of mice with experimental autoimmune encephalomyelitis (EAE).

Minocycline is an oral tetracycline derivative with good bioavailability in the central nervous system (CNS). Minocycline, a potent inhibitor of matrix metalloproteinase (MMP)-9, attenuates disease activity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Potential adverse effects associated with long-term daily minocycline therapy in human patients are concerning. Here, we investigated whether less frequent treatment with long-circulating polyethylene glycol (PEG) minocycline liposomes are effective in treating EAE.

Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.

Laquinimod is an anti-inflammatory agent with good central nervous system (CNS) bioavailability, and neuroprotective and myelorestorative properties. A clinical trial in patients with multiple sclerosis demonstrated that laquinimod significantly reduced loss of brain volume. The cellular substrate or molecular events underlying that treatment effect are unknown. In this study, we aimed to explore laquinimod's potential effects on brain volume, animal behavior, cellular numbers and composition of CNS-intrinsic cells and mononuclear cells within the CNS, amyloid beta (Aβ) accumulation and tau phosphorylation in the F1 3xTg-AD/C3H mouse model of Alzheimer's disease.

The Cre-lox system is a non-dynamic method of gene modification and characterization. Promoters thought to be relatively cell-specific are utilized for generation of cell-lineage-specific gene modifications.

Immunotherapy has the potential to provide a possible treatment therapy to prevent or delay Alzheimer disease. In a clinical trial (AN1792) in which patients received this immunotherapy and received active Aβ1-42 peptide immunizations, treatment was stopped when 6% of patients showed signs of meningoencephalitis. Follow up on these patients led to the conclusion that the antibody response was beneficial in removing Aβ1-42 from brain but an accompanying inflammatory Th1 T cell response was harmful. As a safe alternative treatment targeting the same self protein, Aβ1-42, in brain, we and others are working on a DNA Aβ1-42 immunization protocol as the immune response to DNA immunizations differs in many aspects from immunizations with peptide antigens. Because the immune response to DNA vaccination has different kinetics and has a significantly lower antibody production, we evaluated two different prime boost regimens, Aβ1-42 DNA prime/Aβ1-42 peptide boost and Aβ1-42 peptide prime/Aβ1-42 DNA boost for their effectiveness in antibody production and possible side effects due to inflammatory T cell responses. While both boost regimes significantly enhanced the specific antibody production with comparable antibody concentrations, the absence of the Aβ1-42 T cell response (no proliferation and no cytokine production) is consistent with our previous findings using this DNA Aβ1-42 trimer immunization and greatly enhances the safety aspect for possible clinical use.

Orally available disease-modifying drugs for relapsing-remitting multiple sclerosis (MS) represent an unmet need for this chronic and debilitating disease. Among 5 currently investigated drugs at phase 3 clinical stage, promising efficacy data for fingolimod and oral cladribine have recently been published. However, benefits need to be weighed against the risks to define the role of these compounds within current treatment regimens. In this review, data on the efficacy of a promising compound, oral cladribine, are discussed and balanced with known and anticipated risks in a postmarketing era, and finally gives an outlook on the potential place of this drug in treatment algorithms for MS in the future.

Multiple sclerosis (MS) is thought to be a CD4+ T cell mediated autoimmune demyelinating disease of the central nervous system (CNS) that is rarely diagnosed during infancy. Cellular and molecular mechanisms that confer disease resistance in this age group are unknown. We tested the hypothesis that a differential composition of immune cells within the CNS modulates age-associated susceptibility to CNS autoimmune disease. C57BL/6 mice younger than eight weeks were resistant to experimental autoimmune encephalomyelitis (EAE) following active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35-55. Neonates also developed milder EAE after transfer of adult encephalitogenic T cells primed by adult or neonate antigen presenting cells (APC). There was a significant increase in CD45+ hematopoietic immune cells and CD45+ high side scatter granulocytes in the CNS of adults, but not in neonates. Within the CD45+ immune cell compartment of adults, the accumulation of CD4+ T cells, Gr-1+ and Gr-1- monocytes and CD11c+ dendritic cells (DC) was identified. A significantly greater percentage of CD19+ B cells in the adult CNS expressed MHC II than neonate CNS B cells. Only in the adult CNS could IFNγ transcripts be detected 10 days post immunization for EAE. IFNγ is highly expressed by adult donor CD4+ T cells that are adoptively transferred but not by transferred neonate donor cells. In contrast, IL-17 transcripts could not be detected in adult or neonate CNS in this EAE model, and neither adult nor neonate donor CD4+ T cells expressed IL-17 at the time of adoptive transfer.

The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation.

A brief exposure to systemic hypoxia (i.e., hypoxic preconditioning; HPC) prior to transient middle cerebral artery occlusion (tMCAo) reduces infarct volume, blood-brain barrier disruption, and leukocyte migration. CCL2 (MCP-1), typically regarded as a leukocyte-derived pro-inflammatory chemokine, can also be directly upregulated by hypoxia-induced transcription. We hypothesized that such a hypoxia-induced upregulation of CCL2 is required for HPC-induced ischemic tolerance.

Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 μg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.