Immune monitoring of SARS-CoV-2-specific T cell and B cell responses in patients with multiple sclerosis treated with ocrelizumab.

Introduction: Since the development of the coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there has been significant interest in determining the effectiveness of SARS-CoV-2 vaccines in patients under immunomodulatory or immunosuppressive therapies. The aim of this study was to evaluate the impact of ocrelizumab, a monoclonal anti-CD20 antibody, on SARS-CoV-2-specific T cell and B cell responses in patients with relapsing-remitting multiple sclerosis (RRMS). Methods: To this end, peripheral blood mononuclear cells (PBMCs) were isolated from n = 23 patients with RRMS. Of these patients, n = 17 were tested before (time point t0) and one month after (time point t1) their first dose of ocrelizumab. In addition, we studied n = 9 RRMS patients that got infected with SARS-CoV-2 over the course of ocrelizumab therapy (time point t2). PBMCs were also isolated from n = 19 age- and gender-matched healthy controls (HCs) after vaccination or infection with SARS-CoV-2, respectively. Interferon-γ (IFN-γ)/interleukin-2 (IL-2) and granzyme B (GzB)/perforin (PFN) double-color enzyme-linked immunospot (ELISPOT) assays or single-color ELISPOT assays were performed to measure SARS-CoV-2 antigen-specific T cell and B cell responses. Anti-viral antibody titers were quantified in the serum by chemiluminescence immunoassay. Results: Our data indicate a significant difference in the SARS-CoV-2 specific IFN-γ (P = 0.0119) and PFN (P = 0.0005) secreting T cell compartment in the MS cohort at t0 compared to HCs. Following the first dose of ocrelizumab treatment, a significant decrease in the number of SARS-CoV-2 spike protein-specific B cells was observed (P = 0.0012). Infection with SARS-CoV-2 in MS patients under ocrelizumab therapy did not significantly alter their existing immune response against the virus. Kaplan-Meier survival analysis suggested that the spike S1 protein-specific immunoglobulin (Ig)G response might be a key parameter for predicting the probability of (re)infection with SARS-CoV-2. Discussion: Our results call for a critical discussion regarding appropriate vaccination intervals and potential biomarkers for the prediction of (re)infection with SARS-CoV-2 in patients with MS receiving ocrelizumab. Unique identifier: DRKS00029110; URL: http://apps.who.int/trialsearch/.

A Dual Role of Osteopontin in Modifying B Cell Responses.

The occurrence of B cell aggregates within the central nervous system (CNS) has prompted the investigation of the potential sources of pathogenic B cell and T cell responses in a subgroup of secondary progressive multiple sclerosis (MS) patients. Nevertheless, the expression profile of molecules associated with these aggregates and their role in aggregate development and persistence is poorly described. Here, we focused on the expression pattern of osteopontin (OPN), which is a well-described cytokine, in MS brain tissue. Autopsied brain sections from MS cases with and without B cell pathology were screened for the presence of CD20+ B cell aggregates and co-expression of OPN. To demonstrate the effect of OPN on B cells, flow cytometry, ELISA and in vitro aggregation assays were conducted using the peripheral blood of healthy volunteers. Although OPN was expressed in MS brain tissue independent of B cell pathology, it was also highly expressed within B cell aggregates. In vitro studies demonstrated that OPN downregulated the co-stimulatory molecules CD80 and CD86 on B cells. OPN-treated B cells produced significantly lower amounts of IL-6. However, OPN-treated B cells also exhibited a higher tendency to form homotypic cell aggregates in vitro. Taken together, our data indicate a conflicting role of OPN in modulating B cell responses.

The prevalence of IgG antibodies against milk and milk antigens in patients with multiple sclerosis.

Introduction: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). The pathophysiology of MS is complex and is said to be influenced by multiple environmental determinants, including diet. We and others have previously demonstrated how consumption of bovine milk can aggravate disease severity in MS patients, which can be explained by molecular mimicry between milk antigens and those expressed within the CNS. In this study we set out to identify alternatives to drinking cow milk which might be less detrimental to MS patients who have a genetic predisposition towards developing antibody titers against bovine milk antigens that cross-react with CNS antigens. Methods: To this end, we screened 35 patients with MS and 20 healthy controls for their IgG reactivity against an array of animal-sourced milk, plant-based alternatives as well as individual antigens from bovine milk. Results: We demonstrate that MS patients have a significantly higher IgG response to animal-sourced milk, especially cow milk, in comparison to healthy donors. We also show that the reactivity to cow milk in MS patients can be attributed to reactivity against different bovine milk antigens. Finally, our correlation data indicate the co-existence of antibodies to individual bovine milk antigens and their corresponding cross-reactive CNS antigens. Discussion: Taken together, we suggest screening of blood from MS patients for antibodies against different types of milk and milk antigens in order to establish a personalized diet regimen.

Impact of the Voltage-Gated Calcium Channel Antagonist Nimodipine on the Development of Oligodendrocyte Precursor Cells.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While most of the current treatment strategies focus on immune cell regulation, except for the drug siponimod, there is no therapeutic intervention that primarily aims at neuroprotection and remyelination. Recently, nimodipine showed a beneficial and remyelinating effect in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Nimodipine also positively affected astrocytes, neurons, and mature oligodendrocytes. Here we investigated the effects of nimodipine, an L-type voltage-gated calcium channel antagonist, on the expression profile of myelin genes and proteins in the oligodendrocyte precursor cell (OPC) line Oli-Neu and in primary OPCs. Our data indicate that nimodipine does not have any effect on myelin-related gene and protein expression. Furthermore, nimodipine treatment did not result in any morphological changes in these cells. However, RNA sequencing and bioinformatic analyses identified potential micro (mi)RNA that could support myelination after nimodipine treatment compared to a dimethyl sulfoxide (DMSO) control. Additionally, we treated zebrafish with nimodipine and observed a significant increase in the number of mature oligodendrocytes (* p≤ 0.05). Taken together, nimodipine seems to have different positive effects on OPCs and mature oligodendrocytes.

The presence of cerebellar B cell aggregates is associated with a specific chemokine profile in the cerebrospinal fluid in a mouse model of multiple sclerosis.

BACKGROUND: The presence of meningeal ectopic lymphoid structures (ELS) in a subgroup of patients diagnosed with secondary progressive multiple sclerosis (SPMS) corresponds to a pronounced cortical inflammation and an aggravated disease course. In MP4-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), B cell aggregates develop in the central nervous system (CNS) in the chronic stage of the disease. Therefore, the model is suitable for studying key molecules of ELS development and maintenance. Here, we investigated whether there is a specific cytokine and chemokine signature in paired cerebrospinal fluid (CSF) and serum samples associated with the presence of cerebellar B cell and T cell pathology and B cell aggregates of MP4-immunized mice. METHODS: Paired CSF and serum samples were collected from the cisterna magna and periphery of MP4-immunized mice at the chronic stage of disease. A control group with mice immunized only with the adjuvant (vehicle) was included in the study. A selected panel of 34 cytokines and chemokines were measured by MAGPIX® for both cohorts. For the assessment of B cell and T cell infiltration, immunohistochemical staining was performed and analyzed using light microscopy. To detect specific chemokine receptors additional staining was conducted. RESULTS: While we detected several upregulated cytokines and chemokines in the CSF of MP4-immunized mice independent of the extent of B cell and T cell pathology compared to vehicle-immunized mice, C-C motif chemokine ligand (CCL)-1 was associated with high B cell and T cell infiltration. Furthermore, the level of certain chemokines, including CCL1, CCL5, CCL7, CCL12, CCL22 and C-X-C motif chemokine ligand (CXCL)-13, was significantly increased (p < 0.05) in MP4-immunized mice showing a high number of B cell aggregates. While C-C motif chemokine receptor (CCR)5 had a ubiquitous expression independent of the extent of B cell and T cell pathology, C-X-C motif chemokine receptor (CXCR)-5 and CXCR6 expression was specifically associated with high B cell and T cell pathology. CONCLUSION: Our data suggest that multiple cytokines and chemokines are involved in the pathophysiology of MP4-induced EAE. Furthermore, the presence of B cell aggregates was associated with a specific chemokine profile in the CSF, which might be useful for predicting the presence of these aggregates without the necessity to histologically screen the CNS tissue.

Identification of a novel role for matrix metalloproteinase-3 in the modulation of B cell responses in multiple sclerosis.

There has been a growing interest in the presence and role of B cell aggregates within the central nervous system of multiple sclerosis patients. However, very little is known about the expression profile of molecules associated with these aggregates and how they might be influencing aggregate development or persistence in the brain. The current study focuses on the effect of matrix metalloproteinase-3, which is associated with B cell aggregates in autopsied multiple sclerosis brain tissue, on B cells. Autopsied brain sections from multiple sclerosis cases and controls were screened for the presence of CD20+ B cell aggregates and expression of matrix metalloproteinase-3. Using flow cytometry, enzyme-linked immunosorbent assay and gene array as methods, in vitro studies were conducted using peripheral blood of healthy volunteers to demonstrate the effect of matrix metalloproteinase-3 on B cells. Autopsied brain sections from multiple sclerosis patients containing aggregates of B cells expressed a significantly higher amount of matrix metalloproteinase-3 compared to controls. In vitro experiments demonstrated that matrix metalloproteinase-3 dampened the overall activation status of B cells by downregulating CD69, CD80 and CD86. Furthermore, matrix metalloproteinase-3-treated B cells produced significantly lower amounts of interleukin-6. Gene array data confirmed that matrix metalloproteinase-3 altered the proliferation and survival profiles of B cells. Taken together, out data indicate a role for B cell modulatory properties of matrix metalloproteinase-3.

Antibody cross-reactivity between casein and myelin-associated glycoprotein results in central nervous system demyelination.

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease of the central nervous system (CNS) with a high socioeconomic relevance. The pathophysiology of MS, which is both complex and incompletely understood, is believed to be influenced by various environmental determinants, including diet. Since the 1990s, a correlation between the consumption of bovine milk products and MS prevalence has been debated. Here, we show that C57BL/6 mice immunized with bovine casein developed severe spinal cord pathology, in particular, demyelination, which was associated with the deposition of immunoglobulin G. Furthermore, we observed binding of serum from casein-immunized mice to mouse oligodendrocytes in CNS tissue sections and in culture where casein-specific antibodies induced complement-dependent pathology. We subsequently identified myelin-associated glycoprotein (MAG) as a cross-reactive antigenic target. The results obtained from the mouse model were complemented by clinical data showing that serum samples from patients with MS contained significantly higher B cell and antibody reactivity to bovine casein than those from patients with other neurologic diseases. This reactivity correlated with the B cell response to a mixture of CNS antigens and could again be attributed to MAG reactivity. While we acknowledge disease heterogeneity among individuals with MS, we believe that consumption of cow's milk in a subset of patients with MS who have experienced a previous loss of tolerance to bovine casein may aggravate the disease. Our data suggest that patients with antibodies to bovine casein might benefit from restricting dairy products from their diet.

Effects of a Fully Humanized Type II Anti-CD20 Monoclonal Antibody on Peripheral and CNS B Cells in a Transgenic Mouse Model of Multiple Sclerosis.

Successful therapy with anti-CD20 monoclonal antibodies (mAbs) has reinforced the key role of B cells in the immunopathology of multiple sclerosis (MS). This study aimed to determine the effects of a novel class of anti-CD20 mAbs on vascular and extravascular central nervous system (CNS)-infiltrating B cells in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male hCD20xhIgR3 mice and wild-type C57BL/6 (B6) mice were immunized with human myelin oligodendrocyte glycoprotein (MOG)1-125 to induce EAE. While hCD20xhIgR3 mice were injected intravenously with an anti-human CD20 mAb (5 mg/kg) (rituximab (a type I anti-CD20 mAb) or obinutuzumab (a type II anti-CD20 mAb), B6 mice received the anti-mouse CD20 antibody 18B12. Neither mAb affected clinical disease or serum antibody levels. Obinutuzumab and rituximab had an impact on splenic and CNS-infiltrated B cells with slightly differential depletion efficacy. Additionally, obinutuzumab had beneficial effects on spinal cord myelination. B cell depletion rates in the 18B12/B6 model were comparable with those observed in obinutuzumab-treated hCD20xhIgR3 mice. Our results demonstrate the usefulness of anti-CD20 mAbs for the modulation of B cell-driven peripheral immune response and CNS pathology, with type II antibodies potentially being superior to type I in the depletion of tissue-infiltrating B cells.

Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens.

Multiple sclerosis (MS) has been considered to specifically affect the central nervous system (CNS) for a long time. As autonomic dysfunction including dysphagia can occur as accompanying phenomena in patients, the enteric nervous system has been attracting increasing attention over the past years. The aim of this study was to identify glial and myelin markers as potential target structures for autoimmune processes in the esophagus. RT-PCR analysis revealed glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), and myelin basic protein (MBP) expression, but an absence of myelin oligodendrocyte glycoprotein (MOG) in the murine esophagus. Selected immunohistochemistry for GFAP, PLP, and MBP including transgenic mice with cell-type specific expression of PLP and GFAP supported these results by detection of (1) GFAP, PLP, and MBP in Schwann cells in skeletal muscle and esophagus; (2) GFAP, PLP, but no MBP in perisynaptic Schwann cells of skeletal and esophageal motor endplates; (3) GFAP and PLP, but no MBP in glial cells surrounding esophageal myenteric neurons; and (4) PLP, but no GFAP and MBP in enteric glial cells forming a network in the esophagus. Our results pave the way for further investigations regarding the involvement of esophageal glial cells in the pathogenesis of dysphagia in MS.

B Cells in Multiple Sclerosis and Virus-Induced Neuroinflammation.

Neuroinflammation can be defined as an inflammatory response within the central nervous system (CNS) mediated by a complex crosstalk between CNS-resident and infiltrating immune cells from the periphery. Triggers for neuroinflammation not only include pathogens, trauma and toxic metabolites, but also autoimmune diseases such as neuromyelitis optica spectrum disorders and multiple sclerosis (MS) where the inflammatory response is recognized as a disease-escalating factor. B cells are not considered as the first responders of neuroinflammation, yet they have recently gained focus as a key component involved in the disease pathogenesis of several neuroinflammatory disorders like MS. Traditionally, the prime focus of the role of B cells in any disease, including neuroinflammatory diseases, was their ability to produce antibodies. While that may indeed be an important contribution of B cells in mediating disease pathogenesis, several lines of recent evidence indicate that B cells are multifunctional players during an inflammatory response, including their ability to present antigens and produce an array of cytokines. Moreover, interaction between B cells and other cellular components of the immune system or nervous system can either promote or dampen neuroinflammation depending on the disease. Given that the interest in B cells in neuroinflammation is relatively new, the precise roles that they play in the pathophysiology and progression of different neuroinflammatory disorders have not yet been well-elucidated. Furthermore, the possibility that they might change their function during the course of neuroinflammation adds another level of complexity and the puzzle remains incomplete. Indeed, advancing our knowledge on the role of B cells in neuroinflammation would also allow us to tackle these disorders better. Here, we review the available literature to explore the relationship between autoimmune and infectious neuroinflammation with a focus on the involvement of B cells in MS and viral infections of the CNS.

Antibodies against neural antigens in patients with acute stroke: joint results of three independent cohort studies.

BACKGROUND AND PURPOSE: Ischemic stroke (IS) and hemorrhagic stroke (HemS) typically lead to a breakdown of the blood-brain barrier with neural antigen presentation. This presentation could potentially generate destructive auto-immune responses. Pre-existing antineuronal and antiglial antibodies (AA), predominantly NMDA receptor antibodies, have been reported in patients with stroke. This article summarizes three independent prospective studies, the Lübeck cohort (LC), Barcelona cohort (BC), and Heidelberg cohort (HC), exploring the frequency and clinical relevance of AA in patients with acute stroke (AS). METHODS: In all cohorts together, 344 consecutive patients admitted with AS (322 × IS, 22 × HemS) were screened for AA in serum at admission. Clinical outcome parameters as well as a second AA screening were available at 30 days in the LC or at 90 days in the BC. A control group was included in the BC (20 subjects free from neurological disease) and the HC (78 neurological and ophthalmological patients without evidence for stroke). RESULTS: The rate of positivity for AA was similar in control subjects and AS patients (13%, 95% CI [7%, 22%] vs. 13%, 95% CI [10%, 17%]; p = 0.46) with no significant difference between cohorts (LC 25/171, BC 12/75, HC 9/98). No patient had developed new AA after 30 days, whereas 2 out of 60 patients had developed new AA after 90 days. AA positive patients did not exhibit significant differences to AA negative patients in stroke subtype (LC, BC), initial stroke severity (BC, LC, HC), infarct volume (BC), and functional status at admission (BC, LC, HC) and follow-up (BC, LC). CONCLUSIONS: AS does not induce AA to a relevant degree. Pre-existing AA can be found in the serum of stroke patients, but they do not have a significant association with clinical features and outcomes.