B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

CNS demyelinating events in primary Sjögren's syndrome: A single-center case series on the clinical phenotype.

Objective: The study aimed to assess the prevalence, clinical characteristics, and therapeutic outcomes of the central nervous system (CNS) demyelinating disease in a large cohort of primary Sjögren's syndrome (pSS). Methods: This is an explorative cross-sectional study of patients with pSS seen in the departments of rheumatology, otorhinolaryngology, or neurology of a tertiary university center between January 2015 and September 2021. Results: In a cohort of 194 pSS patients, 22 patients had a CNS manifestation. In this CNS group, 19 patients had a lesion pattern suggestive of demyelination. While there were no obvious differences in the patients' epidemiological disposition or rate of other extraglandular manifestations, the CNS group differed from the remaining patients with pSS by having less glandular manifestations but a higher seroprevalence for anti-SSA/Ro antibodies. Notably, patients with CNS manifestations were often diagnosed with multiple sclerosis (MS) and treated as such, although age and disease course were atypical of MS. Many first-line MS agents were ineffective in these "MS look-alikes"; however, the disease course was benign with B-cell-depleting agents. Conclusion: Neurological symptoms of pSS are common and clinically manifest mainly as myelitis or optic neuritis. Notably, in the CNS, the pSS phenotype can overlap with MS. The prevailing disease is crucial since it has a major impact on the long-term clinical outcome and the choice of disease-modifying agents. Although our observations neither confirm pSS as a more appropriate diagnosis nor rule out simple comorbidity, physicians should consider pSS in the extended diagnostic workup of CNS autoimmune diseases.

Isolation, Culture and Functional Characterization of Glia and Endothelial Cells From Adult Pig Brain.

Primary cultures of glial and endothelial cells are important tools for basic and translational neuroscience research. Primary cell cultures are usually generated from rodent brain although considerable differences exist between human and rodent glia and endothelial cells. Because many translational research projects aim to identify mechanisms that eventually lead to diagnostic and therapeutic approaches to target human diseases, glia, and endothelial cultures are needed that better reflect the human central nervous system (CNS). Pig brain is easily accessible and, in many aspects, close to the human brain. We established an easy and cost-effective method to isolate and culture different primary glial and endothelial cells from adult pig brain. Oligodendrocyte, microglia, astrocyte, and endothelial primary cell cultures were generated from the same brain tissue and grown for up to 8 weeks. Primary cells showed lineage-specific morphology and expressed specific markers with a purity ranging from 60 to 95%. Cultured oligodendrocytes myelinated neurons and microglia secreted tumor necrosis factor alpha when induced with lipopolysaccharide. Endothelial cells showed typical tube formation when grown on Matrigel. Astrocytes enhanced survival of co-cultured neurons and were killed by Aquaporin-4 antibody positive sera from patients with Neuromyelitis optica. In summary, we established a new method for primary oligodendrocyte, microglia, endothelial and astrocyte cell cultures from pig brain that provide a tool for translational research on human CNS diseases.

Interferon-beta specific T cells are associated with the development of neutralizing antibodies in interferon-beta treated multiple sclerosis patients.

Beta-interferons are still among the most commonly used drugs to treat Multiple Sclerosis (MS). The use of beta-interferons is limited by the development of anti-drug antibodies (ADA), which may abrogate the treatment effect of the drug. Although the antibody response has been well studied, little is known about the T cell response to interferon-beta (IFN-ß). We investigated T cell responses in four treatment naïve MS patients and twenty-three patients treated with IFN-ß who had or had not developed ADA to IFN-ß. T cell responses were determined by split-well and primary proliferation assays against different IFN-ß protein preparations and a set of overlapping peptides covering the full sequence of IFN-ß. T cell responses to IFN-ß were observed in all donors. ADA positive patients showed higher T cell responses to IFN-ß protein than ADA negative patients and untreated controls. We identified two immunodominant regions; T cell responses to IFN-ß1-40 were observed in all patients independent of ADA status, while T cell responses to IFN-ß125-159 were stronger in ADA positive than ADA negative patients. IFN-ß specific T cell responses were HLA class II restricted and in ADA positive patients skewed towards a Th2 phenotype. In IFN-ß treated patients we observed a correlation between IFN-ß specific T cell responses, serum ADA titer and loss of biological activity of IFN-ß treatment. Our studies demonstrate the occurrence of an antigen specific HLA class II restricted Th2 T cell response associated with the development of ADA in IFN-ß treated patients.

Quantification and functional characterization of antibodies to native aquaporin 4 in neuromyelitis optica.

BACKGROUND: Antibodies targeting membrane proteins play an important role in various autoimmune diseases of the nervous system. So far, assays allowing proper analysis of such autoantibodies are largely missing. A serum autoantibody to aquaporin 4 (AQP4) is associated with neuromyelitis optica (NMO). Although several assays are able to detect this autoantibody, they do not allow determination of the biological activity of anti-AQP4 antibodies. OBJECTIVE: To develop a bioassay for quantification and characterization of human anti-AQP4 antibodies. DESIGN, SETTING, AND PARTICIPANTS: We developed a novel bioassay for quantification and characterization of human anti-AQP4 antibodies based on high-level expression of native AQP4 (nAQP4) protein on the surface of human astroglioma cells. The test was validated in 2 independent cohorts of patients with NMO spectrum disease. RESULTS: We detected anti-nAQP4-IgG with a sensitivity of 57.9% and specificity of 100% in patients with NMO spectrum diseases, suggesting that our bioassay is at least as sensitive and specific as the gold-standard NMO-IgG assay. The anti-AQP4 antibodies belonged predominantly to the IgG1 isotype and bound with high affinity to the extracellular domain of nAQP4. Our data suggest that the autoantibody exerts pathological properties because nAQP4-IgG-positive sera induced cell death of nAQP4-expressing cells by antibody-dependent cellular natural killer cell cytotoxic effect and complement activation. Furthermore, nAQP4-IgG titers strongly correlated with in vitro cytotoxic effect. CONCLUSIONS: In NMO, this assay may help to unravel the biological function of anti-nAQP4-IgG. Our findings demonstrate the potential of bioassays to characterize biologically relevant antibodies in human autoimmune diseases.

The antidepressant venlafaxine ameliorates murine experimental autoimmune encephalomyelitis by suppression of pro-inflammatory cytokines.

Antidepressants are known to impact on the immune system. In this study, we examined the immunomodulatory properties of venlafaxine, a selective serotonin/norepinephrine reuptake inhibitor (SNRI), in murine experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated CNS demyelinating disease model of multiple sclerosis. EAE was induced in SJL/J mice by adoptive transfer of myelin-specific T cells. Mice received different doses of venlafaxine before induction and after onset of disease. Sustained daily oral treatment with 6, 20 and 60 mg/kg significantly ameliorated the clinical symptoms of the disease compared to vehicle during both preventive and therapeutic intervention. Venlafaxine suppressed the generation of pro-inflammatory cytokines IL-12 p40, TNF-alpha and IFN-gamma in encephalitogenic T-cell clones, splenocytes and peritoneal macrophages in vitro. It also diminished mRNA expression of a number of inflammatory genes in the inflamed CNS tissue, among them CD3, CD8, Granzyme B, IL-12 p40, IFN-gamma, TNF-alpha and the chemokines Ccl2 and RANTES, whereas the expression of brain-derived neurotrophic factor was increased. These findings demonstrate the strong immunomodulatory property of the selective SNRI venlafaxine. Further studies are warranted to clarify whether venlafaxine may exert similar effects in humans.