Björn Tackenberg

Potassium Channel KIR4.1 as an Immune Target in Multiple Sclerosis

BACKGROUND Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system. Many findings suggest that the disease has an autoimmune pathogenesis; the target of the immune response is not yet known. METHODS We screened serum IgG from persons with multiple sclerosis to identify antibodies that are capable of binding to brain tissue and observed specific binding of IgG to glial cells in a subgroup of patients. Using a proteomic approach focusing on membrane proteins, we identified the ATP-sensitive inward rectifying potassium channel KIR4.1 as the target of the IgG antibodies. We used a multifaceted validation strategy to confirm KIR4.1 as a target of the autoantibody response in multiple sclerosis and to show its potential pathogenicity in vivo. RESULTS Serum levels of antibodies to KIR4.1 were higher in persons with multiple sclerosis than in persons with other neurologic diseases and healthy donors (P<0.001 for both comparisons). We replicated this finding in two independent groups of persons with multiple sclerosis or other neurologic diseases (P<0.001 for both comparisons). Analysis of the combined data sets indicated the presence of serum antibodies to KIR4.1 in 186 of 397 persons with multiple sclerosis (46.9%), in 3 of 329 persons with other neurologic diseases (0.9%), and in none of the 59 healthy donors. These antibodies bound to the first extracellular loop of KIR4.1. Injection of KIR4.1 serum IgG into the cisternae magnae of mice led to a profound loss of KIR4.1 expression, altered expression of glial fibrillary acidic protein in astrocytes, and activation of the complement cascade at sites of KIR4.1 expression in the cerebellum. CONCLUSIONS KIR4.1 is a target of the autoantibody response in a subgroup of persons with multiple sclerosis. (Funded by the German Ministry for Education and Research and Deutsche Forschungsgemeinschaft.).

Impaired inhibitory Fcγ receptor IIB expression on B cells in chronic inflammatory demyelinating polyneuropathy

The inhibitory Fc-γ receptor FcγRIIB, expressed on myeloid and B cells, has a critical role in the balance of tolerance and autoimmunity, and is required for the antiinflammatory activity of intravenous Ig (IVIG) in various murine disease models. However, the function of FcγRIIB and its regulation by IVIG in human autoimmune diseases are less well understood. Chronic inflammatory demyelinating polyneuropathy (CIDP) is the most common treatable acquired chronic polyneuropathy, and IVIG is widely used as a first-line initial and maintenance treatment. We found that untreated patients with CIDP, compared with demographically matched healthy controls, showed consistently lower FcγRIIB expression levels on naive B cells, and failed to up-regulate or to maintain up-regulation of FcγRIIB as B cells progressed from the naive to the memory compartment. Concomitantly, the rare −386C/−120A FcγRIIB promoter polymorphism resulting in reduced promoter activity previously associated with autoimmune phenotypes was overrepresented in CIDP. Also, FcγRIIB protein expression was up-regulated on monocytes and B cells after clinically effective IVIG therapy. Thus, our results suggest that the inhibitory FcγRIIB is impaired at a critical B cell differentiation checkpoint in CIDP, and that modulating FcγRIIB expression might be a promising approach to efficiently limit antibody-mediated immunopathology in CIDP.

EBNA1-specific T cells from patients with multiple sclerosis cross react with myelin antigens and co-produce IFN-γ and IL-2

Symptomatic primary Epstein-Barr virus (EBV) infection and elevated humoral immune responses to EBV are associated with an increased risk of developing multiple sclerosis (MS). We explored mechanisms leading to this change in EBV-specific immunity in untreated patients with MS and healthy virus carriers matched for MS-associated HLA alleles. MS patients showed selective increase of T cell responses to the EBV nuclear antigen 1 (EBNA1), the most consistently recognized EBV-derived CD4+ T cell antigen in healthy virus carriers, but not to other EBV-encoded proteins. In contrast, influenza and human cytomegalovirus–specific immune control was unchanged in MS. The enhanced response to EBNA1 was mediated by an expanded reservoir of EBNA1-specific central memory CD4+ T helper 1 (Th1) precursors and Th1 (but not Th17) polarized effector memory cells. In addition, EBNA1-specific T cells recognized myelin antigens more frequently than other autoantigens that are not associated with MS. Myelin cross-reactive T cells produced IFN-γ, but differed from EBNA1-monospecific cells in their capability to produce interleukin-2, indicative of a polyfunctional phenotype as found in controlled chronic viral infections. Our data support the concept that clonally expanded EBNA1-specific CD4+ T cells potentially contribute to the development of MS by cross-recognition of myelin antigens.

Cytokines and epilepsy

Epilepsy is a common chronic neurological disorder affecting approximately 8 out of 1000 people. Its pathophysiology, however, has remained elusive in many regards. Consequently, adequate seizure control is still lacking in about one third of patients. Cytokines are soluble mediators of cell communication that are critical in immune regulation. In recent years, studies have shown that epileptic seizures can induce the production of cytokines, which in turn influence the pathogenesis and course of epilepsies. At the time of this review, the focus is mostly on interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα). In this review, we summarize the current knowledge regarding these cytokines and their potential roles in epilepsy. The focus concentrates on their expression and influence on induced seizures in animal models of epilepsy, as well as findings in human studies. Both proconvulsive and anticonvulsive effects have been reported for each of these molecules. One possible explanation for this phenomenon is that cytokines play dichotomous roles through multiple pathways, each of which is dependent on free concentration and available receptors. Furthermore, the immune-mediated leakage in the blood-brain-barrier also plays an important role in epileptogenesis. Nonetheless, these observations demonstrate the multifarious nature of cytokine networks and the complex relationship between the immune system and epilepsy. Future studies are warranted to further clarify the influence of the immune system on epilepsy and vice versa.

Neurofascin as a target for autoantibodies in peripheral neuropathies

ABSTRACT Objectives: We asked whether autoantibodies against neurofascin (NF)186 or NF155, both localized at the nodes of Ranvier, are present in serum of patients with inflammatory neuropathy, and whether NF-specific monoclonal antibodies are pathogenic in vivo. Methods: We cloned human NF155 and NF186, and developed an ELISA and cell-based assay to screen for antibodies to human NF in a total of 434 donors including 294 patients with Guillain-Barré syndrome variants acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy, and chronic inflammatory demyelinating polyneuropathy (CIDP). We characterized reactive samples by isotyping, tissue section staining, and epitope mapping. We also injected NF-specific monoclonal antibodies IV into rats with experimental autoimmune neuritis. Results: We detected autoantibodies to NF by ELISA in 4% of patients with AIDP and CIDP, but not in controls. Most positive samples contained immunoglobulin G (IgG)1, IgG3, or IgG4 antibodies directed to only one isoform of NF. Two patients with CIDP showed particularly high (1:10,000 dilution) NF155-specific reactivity in both assays and stained paranodes. Two other patients with CIDP who benefited from plasma exchange exhibited antibodies to NF155 by ELISA, and upon affinity purification, antibodies to both isoforms were observed by both assays. Anti-NF monoclonal antibodies enhanced and prolonged induced neuritis in rats. Conclusions: Autoantibodies to NF are detected in a very small proportion of patients with AIDP and patients with CIDP, but may nevertheless be pathogenic in these cases.

Risk for Myasthenia Gravis Maps to a (151)Pro -> Ala Change in TNIP1 and to Human Leukocyte Antigen-B*08

The objective of this study is to comprehensively define the genetic basis of early onset myasthenia gravis (EOMG).

Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity

IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

Clonal expansions of CD4+ B helper T cells in autoimmune myasthenia gravis.

The weakness in myasthenia gravis (MG) is mediated by T helper cell (Th)‐dependent autoantibodies against neuromuscular epitopes. So far, analyzing Th phenotypes or antigen specificities has yielded very few clues to pathogenesis. Here we adopt an alternative antigen‐independent approach, analyzing T cell receptor (TCR) Vβ usage/expansions in blood from 118 MG patients. We found major expansions (⩾ five standard deviations above the mean of 118 healthy, individually age‐ and sex‐matched controls) in diverse Vβ in 21 patients (17.6%, p<0.001) among CD4+ T cells, and in 45 patients (38.1%, p<0.001) among CD8+ T cells. In informative probands, the expanded CD4+ cells consistently showed a Th cell phenotype (CD57+CXCR5+) and expressed Th1 cytokines. Furthermore, their expression of markers for activation, lymphocyte trafficking and B cell‐activating ability persisted for ⩾3 years. Surprisingly, we noted a selective decline in the expansions/their CD57 positivity while the probands’ MG was improving. CDR3 spectratyping suggested mono‐ or oligoclonal origins, which were confirmed by the prevalent TCR Vβ CDR3 sequences of Th cells cloned from repeat bleeds. Thus, our data provide evidence for persistent clonally expanded CD4+ B helper T cell populations in the blood of MG patients. These unexpected CD4+ expansions might hold valuable clues to MG immunopathogenesis.

Interictal alterations of cytokines and leukocytes in patients with active epilepsy.

BACKGROUND Involvement of the innate immune system in the pathogenesis of epilepsies has been suggested but possible interactions between the immune system and human epilepsy remain unclear. We analyzed the interictal immuno-phenotype of leukocyte subsets and proinflammatory cytokine profiles in epileptic patients and correlated them with the epilepsy syndrome. METHODS 101 patients with active focal or generalized epilepsy were prospectively included and compared to 36 healthy controls. Immuno-phenotype of leukocyte subsets and cytokines IL-1β, IL-6 and tnfα were measured in peripheral blood. Multivariate analyses were performed to test group differences. RESULTS As compared to controls, the patients showed an elevated percentage of monocytes (18.06±7.08% vs. 12.68±4.55%, p<0.001), NK cells (14.88±7.08% vs. 11.43±5.41%, p=0.019) and IL-6 concentration (3.33±3.11 pg/ml vs. 1.5±1.36 pg/ml, p=0.002). This remained true when focal epilepsies or generalized epilepsies were compared separately to controls but only focal epilepsies showed additionally a decrease in B lymphocyts (8.16±3.76% vs. 11.54±4.2%, p<0.001). Treatment with lamotrigine was associated with a higher percentage of B lymphocytes and valproate with an increased percentage of CD4(+) T lymphocytes. Therapy with levetiracetam showed a trend towards decreased CD8(+) T cell counts. No significant differences were seen between focal and generalized epilepsies and between temporal and extratemporal lobe epilepsies. CONCLUSION Patients with active epilepsy revealed interictal alterations of the immune system which varied among specific syndromes and were influenced by antiepileptic drug treatment.

PML risk stratification using anti-JCV antibody index and L-selectin.

Background: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters. Objective: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification. Methods: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients). Results: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor “CD62L low” increasing a patient’s relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group. Conclusions: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence.