Marie Wunsch

Identification of a B cell-dependent subpopulation of multiple sclerosis by measurements of brain-reactive B cells in the blood

B cells are increasingly coming into play in the pathogenesis of multiple sclerosis (MS). Here, we screened peripheral blood mononuclear cells (PBMC) from patients with clinically isolated syndrome (CIS), MS, other non-inflammatory neurological, inflammatory neurological or autoimmune diseases, and healthy donors for their B cell reactivity to CNS antigen using the enzyme-linked immunospot technique (ELISPOT) after 96 h of polyclonal stimulation. Our data show that nine of 15 patients with CIS (60.0%) and 53 of 67 patients with definite MS (79.1%) displayed CNS-reactive B cells, compared to none of the control donors. The presence of CNS-reactive B cells in the blood of the majority of patients with MS or at risk to develop MS along with their absence in control subjects suggests that they might be indicative of a B cell-dependent subpopulation of the disease.

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity.

Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the TH1, TH2, and TH17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospot® assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)-γ and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of TH1, TH2, and TH17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN-γ and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the TH1, TH2, and TH17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.

Nimodipine fosters remyelination in a mouse model of multiple sclerosis and induces microglia-specific apoptosis

Significance Multiple sclerosis (MS) is the most frequent neurological disease that leads to premature retirement in young adults. Progressive MS currently is not only incurable, but also untreatable. Here we show that the calcium channel antagonist nimodipine significantly attenuated clinical disease and central nervous system degeneration and also fostered remyelination in a mouse model of MS. The effect of nimodipine was microglia specific, inducing apoptosis and decreasing the production of neurotoxic molecules such as nitric oxide and reactive oxygen species both in vitro and in vivo. These results introduce a treatment option for MS and also may have broad therapeutic implications for chronic neuroinflammatory diseases in general. Despite continuous interest in multiple sclerosis (MS) research, there is still a lack of neuroprotective strategies, because the main focus has remained on modulating the immune response. Here we performed in-depth analysis of neurodegeneration in experimental autoimmune encephalomyelitis (EAE) and in in vitro studies regarding the effect of the well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated clinical EAE and spinal cord degeneration and promoted remyelination. Surprisingly, we observed calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell-type specific and irrespective of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS and reactive oxygen species levels in EAE. In addition, increased numbers of Olig2+APC+ oligodendrocytes were detected. Overall, nimodipine application seems to generate a favorable environment for regenerative processes and therefore could be a treatment option for MS, because it combines features of immunomodulation with beneficial effects on neuroregeneration.

Differential effects of FTY720 on the B cell compartment in a mouse model of multiple sclerosis

BackgroundMP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS).MethodsMP4-immunized mice were treated orally with FTY720 for 30xa0days at the peak of disease or 50xa0days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro.ResultsFTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs.ConclusionsThe data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE.

Central nervous system infiltrates are characterized by features of ongoing B cell-related immune activity in MP4-induced experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS) lymphoid follicle-like aggregates have been reported in the meninges of patients. Here we investigated the functional relevance of B cell infiltration into the central nervous system (CNS) in MP4-induced experimental autoimmune encephalomyelitis (EAE), a B cell-dependent mouse model of MS. In chronic EAE, B cell aggregates were characterized by the presence of CXCL13(+) and germinal center CD10(+) B cells. Germline transcripts were expressed in the CNS and particularly related to TH17-associated isotypes. We also observed B cells with restricted VH gene usage that differed from clones found in the spleen. Finally, we detected CNS-restricted spreading of the antigen-specific B cell response towards a myelin and a neuronal autoantigen. These data imply the development of autonomous B cell-mediated autoimmunity in the CNS in EAE - a concept that might also apply to MS itself.

KIR4.1 Antibodies as Biomarkers in Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disease caused by the infiltration of autoreactive lymphocytes into the central nervous system (CNS). The influence of B cells has been underestimated for a long time. Recently, it has become more apparent that B cells can fundamentally contribute to the pathogenesis of MS in terms of antigen presentation, co-stimulation, cytokine production, ectopic lymphoneogenesis, and antibody secretion (1–3). Along these lines, B cell depletion in MS patients with a relapsing-remitting disease course was able to reduce inflammatory brain lesions and clinical relapses (4) and B cell activation influences T cell polarization in the animal model of MS (5). Great effort has been made to utilize B cells for dividing patients into subgroups and to predict treatment responses in individual patients to specific drugs (6). Especially the detection of autoantibodies against CNS antigens is a crucial research subject due to its capability to be used as disease-specific biomarkers for the diagnosis and prognosis of MS. n nTo this end, in a clinical study published in 2012 in the New England Journal of Medicine, Srivastava and colleagues screened serum samples aiming to identify CNS-specific antibodies in MS (7). The authors identified the glial potassium channel KIR4.1 as one of the serum antibody targets in MS patients. Subsequently, an enzyme-linked immunosorbent assay (ELISA) large-scale screening with 397 MS patients, 329 persons with other neurological diseases, and 59 healthy donors was performed. Antibodies against KIR4.1 were observed in 46.9% of patients with MS, but were essentially absent in people with other neurological diseases and healthy donors. Based on these data, Srivastava et al. concluded that KIR4.1 is a CNS-specific target of the autoantibody response in a subgroup of patients with MS. n nWe would like to carefully raise the possibility that the serum IgG reactivity against KIR4.1 might present an epiphenomenon and there could be an additional target for autoreactive B cells beyond the CNS. It has to be considered that KIR4.1 is not only expressed in the CNS, but also on parietal cells of the gastric mucosal epithelium (8). The KIR4.1 channel expression is essential for the parietal cell control of acid secretion (9, 10). It is well known that there is a connection between the gastrointestinal immune response and CNS autoimmunity (11, 12). There are several possible mechanisms including molecular mimicry, determinant spreading, or bystander activation explaining the link between gastrointestinal immune response and MS (13). As recently published by Banati et al. (11), antibodies against parietal cells are also present in the sera of patients with MS. The presence of anti-parietal cell antibodies was most frequently associated with gastrointestinal complaints of the MS patients (11). Thus, it is possible that an impaired gastric mucosa, which can appear in cases of a frequent corticosteroid therapy, could extensively expose the parietal cell KIR4.1 to B cells. Corticosteroids augment gastroduodenal permeability and high doses are associated with macroscopic mucosal lesions (14). n nTherefore, we consider it as necessary to include the history of gastrointestinal complaints in the anamnesis of the study subjects before defining KIR4.1 as a CNS target of serum antibodies. In the setting of patients with an initial demyelinating event who have not received corticosteroids, one could readily exclude an impact of the MS medication on the gut. Finding anti-KIR4.1 antibodies in such patients in combination with gut impairments could raise the possibility that the gut itself is also a target of the autoimmune process. n nWhile further investigations are awaited to clearly analyze the impact of immune responses against antigens of gastric parietal cells on the finding of KIR4.1-reactive antibodies in patients with MS, the diagnostic potential of serum KIR4.1 antibodies may be highly clinically relevant. Therefore, the sensitivity and specificity of the initial findings of serum KIR4.1 antibodies in a subgroup of MS patients should be confirmed in studies from independent researchers. n nFurthermore, the pathogenicity of KIR4.1 antibodies needs to be clarified. Only 2 of 19 serum IgG positive MS patients also had detectable levels of KIR4.1 antibodies in the cerebrospinal fluid (CSF) (7). By analogy, aquaporin-4 antibodies were not readily detected in the CSF in neuromyelitis optica patients (15). Immunhistochemical investigations by Lennon et al. (16) showed that serum aquaporin-4 antibodies in patients with neuromyelitis optica bind to CNS tissue as well as to parietal cells of the gastric mucosa (16). However, it is beyond doubt that aquaporin-4 antibodies are highly pathogenic in neuromyelitis optica since they were studied extensively. Kinoshita and colleagues have demonstrated the pathogenic role of aquaporin-4 antibodies in vivo (17). They obtained serum IgG from patients with neuromyelitis optica and detected astrocytic damage in rats after passive transfer of aquaporin-4 containing IgG. In vivo experiments of this kind could delineate whether KIR4.1 antibodies are only a disease marker or if they play a central pathogenic role. To this end, KIR4.1 antibody containing serum could be injected into mice and subsequent histological and immunohistochemical investigations could show potential resulting pathological effects in CNS and gut. Furthermore, astrocytes should be incubated with KIR4.1 antibody containing serum and cell viability assays may demonstrate whether KIR4.1-reactive IgG induces astrocytic cytotoxicity.

The enteric nervous system is a potential autoimmune target in multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.

The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis

There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.

Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay

As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.

CEACAM1 mediates B cell aggregation in central nervous system autoimmunity

B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1+ B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines, application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain −3 (TIM-3) on B cells, a novel molecule that has recently been described to induce anergy in T cells. Interestingly, elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall, these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease.