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

High-Dose Vitamin D-Mediated Hypercalcemia as a Potential Risk Factor in Central Nervous System Demyelinating Disease

The exact cause of multiple sclerosis (MS) is unknown; however, it is considered to be an inflammatory disease of the central nervous system (CNS) triggered by a combination of both environmental and genetic factors. Vitamin D deficiency is also discussed as a possible disease-promoting factor in MS, as low vitamin D status is associated with increased formation of CNS lesions, elevated number of relapses and accelerated disease progression. However, it remains unclear whether this association is causal and related and most importantly, whether vitamin D supplementation in MS is of direct therapeutic benefit. Recently, we could show that in a murine model of MS, administration of a moderate vitamin D dose was of clinical benefit, while excessive vitamin D supplementation had a negative effect on disease severity. Of note, disease exacerbation was associated with high-dose vitamin D caused secondary hypercalcemia. Mechanistically dissecting this outcome, we found that hypercalcemia independent of vitamin D similarly triggered activation of disease-perpetuating T cells. These findings caution that vitamin D should be supplemented in a controlled and moderate manner in patients with MS and concomitantly highlight calcium as a novel potential MS risk factor by itself. In this review, we will summarize the current evidence from animal and clinical studies aiming to assess whether vitamin D may be of benefit in patients with MS. Furthermore, we will discuss any possible secondary effects of vitamin D with a particular focus on the role of calcium on immune cells and in the pathogenesis of CNS demyelinating disease

Targeting B Cells and Microglia in Multiple Sclerosis With Bruton Tyrosine Kinase Inhibitors

ImportanceCurrently, disease-modifying therapies for multiple sclerosis (MS) use 4 mechanisms of action: immune modulation, suppressing immune cell proliferation, inhibiting immune cell migration, or cellular depletion. Over the last decades, the repertoire substantially increased because of the conceptual progress that not only T cells but also B cells play an important pathogenic role in MS, fostered by the empirical success of B cell–depleting antibodies against the surface molecule CD20. Notwithstanding this advance, a continuous absence of B cells may harbor safety risks, such as a decline in the endogenous production of immunoglobulins. Accordingly, novel B cell–directed MS therapies are in development, such as inhibitors targeting Bruton tyrosine kinase (BTK).ObservationsBTK is centrally involved in the B cell receptor–mediated activation of B cells, one key requirement in the development of autoreactive B cells, but also in the activation of myeloid cells, such as macrophages and microglia. Various compounds in development differ in their binding mode, selectivity and specificity, relative inhibitory concentration, and potential to enter the central nervous system. The latter may be important in assessing whether BTK inhibition is a promising strategy to control inflammatory circuits within the brain, the key process that is assumed to drive MS progression. Accordingly, clinical trials using BTK inhibitors are currently conducted in patients with relapsing-remitting MS as well as progressive MS, so far generating encouraging data regarding efficacy and safety.Conclusions and RelevanceWhile the novel approach of targeting BTK is highly promising, several questions remain unanswered, such as the long-term effects of using BTK inhibitors in the treatment of inflammatory CNS disease. Potential changes in circulating antibody levels should be evaluated and compared with B cell depletion. Also important is the potential of BTK inhibitors to enter the CNS, which depends on the given compound. Remaining questions involve where BTK inhibitors fit in the landscape of MS therapeutics. A comparative analysis of their distinct properties is necessary to identify which inhibitors may be used in relapsing vs progressive forms of MS as well as to clarify which agent may be most suitable for sequential use after anti-CD20 treatment.</jats:sec

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

Abstract 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.EMD Serono http://dx.doi.org/10.13039/10000475