Antibody-mediated initiation and lymphocyte-targeting therapies in CNS demyelinating disease

Multiple sclerosis is inflammatory central nervous system disease, characterised by areas of demyelination and axonal loss. The pathogenic mechanism behind the disease still remains unknown, however it is thought to be mainly T cell-mediated. Notwithstanding this, B cells have increasingly been recognized as key mediators of disease. This work focuses on three distinct characteristics of MS pathology in the animal model experimental autoimmune encephalomyelitis (EAE). The first project focused on establishing inhibition of Bruton´s tyrosine kinase (BTK) as a novel therapeutic approach. BTK is centrally placed in B cell receptor (BCR) signalling. In a B cell-mediated EAE model, induced by injection of MOG protein, we observed that evobrutinib, a novel BTK inhibitor, dose-dependently reduced clinical disease. Evobrutinib inhibited BCR-mediated phenotypic maturation of B cells from follicular (FO) II to FO I and reduced activation of B cells and T cells. It diminished calcium mobilization and cytokine production after BCR stimulation in murine and human B cells. Investigating MS patients, we did not observe a difference in B cell frequency, BTK expression or phosphorylation of BTK after BCR stimulation. Taken together, we demonstrated that BTK inhibition (BTKi) is a promising new strategy to control pathogenic B cell activity in a model of CNS autoimmunity. The second project investigated the effects of long-term high dose vitamin D supplementation on the peripheral immune system and EAE severity. We observed clinical and histological deterioration of EAE after long-term high dose supplementation of vitamin D. Further investigations traced this effect to a secondary hypercalcemia, which in contrast to vitamin D, increased the activation and differentiation of T cells both in vitro and in vivo. Since MS patients are often continuously supplemented with vitamin D over long periods of time, our work cautions patients and clinicians to be attentive of potential side effects by hypercalcemia. The third project focused on antibodies in the initiation of disease. We investigated the capacity of myelin-reactive antibodies to facilitate encephalitogenic responses via opsonisation of CNS antigen. We observed that antibody production in the absence of B cells was sufficient to induce EAE in a transgenic mouse model. Additionally, adoptive transfer of antibodies in mice containing MOG-specific T cells induced disease via otherwise unresponsive myeloid antigen-presenting cells (APCs). MOG-targeting antibodies enabled Fcreceptor (FcR) mediated recognition and phagocytosis in in vitro differentiated macrophages. Additionally, antibody preparations from neuromyelitis optica patients positive for MOG antibodies similarly facilitated recognition by myeloid APCs. These results establish opsonisation of CNS antigen by specific antibodies as a novel mechanism to trigger CNS demyelination.2020-07-0

Functional characterization of reappearing B cells after anti-CD20 treatment of CNS autoimmune disease.

The anti-CD20 antibody ocrelizumab, approved for treatment of multiple sclerosis, leads to rapid elimination of B cells from the blood. The extent of B cell depletion and kinetics of their recovery in different immune compartments is largely unknown. Here, we studied how anti-CD20 treatment influences B cells in bone marrow, blood, lymph nodes, and spleen in models of experimental autoimmune encephalomyelitis (EAE). Anti-CD20 reduced mature B cells in all compartments examined, although a subpopulation of antigen-experienced B cells persisted in splenic follicles. Upon treatment cessation, CD20+ B cells simultaneously repopulated in bone marrow and spleen before their reappearance in blood. In EAE induced by native myelin oligodendrocyte glycoprotein (MOG), a model in which B cells are activated, B cell recovery was characterized by expansion of mature, differentiated cells containing a high frequency of myelin-reactive B cells with restricted B cell receptor gene diversity. Those B cells served as efficient antigen-presenting cells (APCs) for activation of myelin-specific T cells. In MOG peptide-induced EAE, a purely T cell-mediated model that does not require B cells, in contrast, reconstituting B cells exhibited a naive phenotype without efficient APC capacity. Our results demonstrate that distinct subpopulations of B cells differ in their sensitivity to anti-CD20 treatment and suggest that differentiated B cells persisting in secondary lymphoid organs contribute to the recovering B cell pool

Inhibition of Bruton’s tyrosine kinase interferes with pathogenic B-cell development in inflammatory CNS demyelinating disease

Anti-CD20-mediated B-cell depletion effectively reduces acute multiple sclerosis (MS) flares. Recent data shows that antibody-mediated extinction of B cells as a lasting immune suppression, harbors the risk of developing humoral deficiencies over time. Accordingly, more selective, durable and reversible B-cell-directed MS therapies are needed. We here tested inhibition of Bruton's tyrosine kinase (BTK), an enzyme centrally involved in B-cell receptor signaling, as the most promising approach in this direction. Using mouse models of MS, we determined that evobrutinib, the first BTK inhibiting molecule being developed, dose-dependently inhibited antigen-triggered activation and maturation of B cells as well as their release of pro-inflammatory cytokines. Most importantly, evobrutinib treatment functionally impaired the capacity of B cells to act as antigen-presenting cells for the development of encephalitogenic T cells, resulting in a significantly reduced disease severity in mice. In contrast to anti-CD20, BTK inhibition silenced this key property of B cells in MS without impairing their frequency or functional integrity. In conjunction with a recent phase II trial reporting that evobrutinib is safe and effective in MS, our mechanistic data highlight therapeutic BTK inhibition as a landmark towards selectively interfering with MS-driving B-cell properties

High dose vitamin D exacerbates central nervous system autoimmunity by raising T-cell excitatory calcium

Poor vitamin D status is associated with a higher relapse rate and earlier disability in multiple sclerosis. Based on these associations, patients with multiple sclerosis are frequently supplemented with the vitamin D precursor cholecalciferol, although it is unclear whether this regimen is of therapeutic benefit. To model consequences of this common practice, mice were fed for more than 3 months with a low, medium or high dose of cholecalciferol, representative of vitamin D deficiency, modest and disproportionally high supplementation, respectively, in patients with multiple sclerosis. Compared to vitamin D-deprived mice, its moderate supplementation reduced the severity of subsequent experimental autoimmune encephalomyelitis, which was associated with an expansion of regulatory T cells. Direct exposure of murine or human T cells to vitamin D metabolites inhibited their activation. In contrast, mice with 25-(OH) vitamin D levels above 200 nmol/l developed fulminant experimental autoimmune encephalomyelitis with massive CNS infiltration of activated myeloid cells, Th1 and Th17 cells. When dissecting this unexpected outcome, we observed that high, but not medium dose vitamin D had caused mild hypercalcaemia, which rendered T cells more prone to pro-inflammatory activation. Exposing murine or human T cells to equivalent calcium concentrations in vitro enhanced its influx, triggering activation, upregulation of pro-inflammatory gene products and enhanced transmigration across a blood-brain barrier model. These findings suggest that vitamin D at moderate levels may exert a direct regulatory effect, while continuous high dose vitamin D treatment could trigger multiple sclerosis disease activity by raising mean levels of T-cell excitatory calcium

Functional characterization of reappearing B cells after anti-CD20 treatment of CNS autoimmune disease.

The anti-CD20 antibody ocrelizumab, approved for treatment of multiple sclerosis, leads to rapid elimination of B cells from the blood. The extent of B cell depletion and kinetics of their recovery in different immune compartments is largely unknown. Here, we studied how anti-CD20 treatment influences B cells in bone marrow, blood, lymph nodes, and spleen in models of experimental autoimmune encephalomyelitis (EAE). Anti-CD20 reduced mature B cells in all compartments examined, although a subpopulation of antigen-experienced B cells persisted in splenic follicles. Upon treatment cessation, CD20+ B cells simultaneously repopulated in bone marrow and spleen before their reappearance in blood. In EAE induced by native myelin oligodendrocyte glycoprotein (MOG), a model in which B cells are activated, B cell recovery was characterized by expansion of mature, differentiated cells containing a high frequency of myelin-reactive B cells with restricted B cell receptor gene diversity. Those B cells served as efficient antigen-presenting cells (APCs) for activation of myelin-specific T cells. In MOG peptide-induced EAE, a purely T cell-mediated model that does not require B cells, in contrast, reconstituting B cells exhibited a naive phenotype without efficient APC capacity. Our results demonstrate that distinct subpopulations of B cells differ in their sensitivity to anti-CD20 treatment and suggest that differentiated B cells persisting in secondary lymphoid organs contribute to the recovering B cell pool

Myelin-reactive antibodies initiate T cell-mediated CNS autoimmune disease by opsonization of endogenous antigen

In the pathogenesis of central nervous system (CNS) demyelinating disorders, antigen-specific B cells are implicated to act as potent antigen-presenting cells (APC), eliciting waves of inflammatory CNS infiltration. Here, we provide the first evidence that CNS-reactive antibodies (Ab) are similarly capable of initiating an encephalitogenic immune response by targeting endogenous CNS antigen to otherwise inert myeloid APC. In a transgenic mouse model, constitutive production of Ab against myelin oligodendrocyte glycoprotein (MOG) was sufficient to promote spontaneous experimental autoimmune encephalomyelitis (EAE) in the absence of B cells, when mice endogenously contained MOG-recognizing T cells. Adoptive transfer studies corroborated that anti-MOG Ab triggered activation and expansion of peripheral MOG-specific T cells in an Fc-dependent manner, subsequently causing EAE. To evaluate the underlying mechanism, anti-MOG Ab were added to a co-culture of myeloid APC and MOG-specific T cells. At otherwise undetected concentrations, anti-MOG Ab enabled Fc-mediated APC recognition of intact MOG; internalized, processed and presented MOG activated naïve T cells to differentiate in an encephalitogenic manner. In a series of translational experiments, anti-MOG Ab from two patients with an acute flare of CNS inflammation likewise facilitated detection of human MOG. Jointly, these observations highlight Ab-mediated opsonization of endogenous CNS auto-antigen as a novel disease- and/or relapse-triggering mechanism in CNS demyelinating disorders