Kevin C. O'Connor

Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein

The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.

Dysregulated T cell expression of TIM3 in multiple sclerosis

T cell immunoglobulin- and mucin domain–containing molecule (TIM)3 is a T helper cell (Th)1–associated cell surface molecule that regulates Th1 responses and promotes tolerance in mice, but its expression and function in human T cells is unknown. We generated 104 T cell clones from the cerebrospinal fluid (CSF) of six patients with multiple sclerosis (MS) (n = 72) and four control subjects (n = 32) and assessed their cytokine profiles and expression levels of TIM3 and related molecules. MS CSF clones secreted higher amounts of interferon (IFN)-γ than did those from control subjects, but paradoxically expressed lower levels of TIM3 and T-bet. Interleukin 12–mediated polarization of CSF clones induced substantially higher amounts of IFN-γ secretion but lower levels of TIM3 in MS clones relative to control clones, demonstrating that TIM3 expression is dysregulated in MS CSF clones. Reduced levels of TIM3 on MS CSF clones correlated with resistance to tolerance induced by costimulatory blockade. Finally, reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-γ secretion, directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-γ secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel, intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases.

B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes

In multiple sclerosis patients, B cells mature in the draining cervical lymph nodes before trafficking across the blood-brain barrier. B Cells Flip the Switch for MS B cells in multiple sclerosis (MS) patients may mature outside the central nervous system (CNS). Two complementary studies report that B cells found outside the CNS—in peripheral blood and draining cervical lymph nodes (CLNs)—share antigen specificity with intrathecal B cell repertoires. In patients with MS, immune cells attack the CNS; however, it remains unclear whether these cells mature in the CNS or traffic to the CNS as mature cells. Using paired tissues and high-throughput sequencing, Stern et al. found that clonally expanded B cells are found in both the CNS and CLNs but that founding members were more often found in the draining CLNs. Palanichamy et al. extend these findings by reporting a peripheral blood/CNS axis of mature B cells that have undergone class switch. These data support the therapeutic use of monoclonal antibodies that prevent lymphocytes from crossing the blood-brain barrier or induce peripheral B cell depletion in MS patients. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by autoimmune-mediated demyelination and neurodegeneration. The CNS of patients with MS harbors expanded clones of antigen-experienced B cells that reside in distinct compartments including the meninges, cerebrospinal fluid (CSF), and parenchyma. It is not understood whether this immune infiltrate initiates its development in the CNS or in peripheral tissues. B cells in the CSF can exchange with those in peripheral blood, implying that CNS B cells may have access to lymphoid tissue that may be the specific compartment(s) in which CNS-resident B cells encounter antigen and experience affinity maturation. Paired tissues were used to determine whether the B cells that populate the CNS mature in the draining cervical lymph nodes (CLNs). High-throughput sequencing of the antibody repertoire demonstrated that clonally expanded B cells were present in both compartments. Founding members of clones were more often found in the draining CLNs. More mature clonal members derived from these founders were observed in the draining CLNs and also in the CNS, including lesions. These data provide new evidence that B cells traffic freely across the tissue barrier, with the majority of B cell maturation occurring outside of the CNS in the secondary lymphoid tissue. Our study may aid in further defining the mechanisms of immunomodulatory therapies that either deplete circulating B cells or affect the intrathecal B cell compartment by inhibiting lymphocyte transmigration into the CNS.

Age-Dependent B Cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis

Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface Ags in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. Although Abs to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum Abs that brightly labeled the MOG transfectant. Abs to two other myelin surface Ags were largely absent. Affinity purification of MOG Abs as well as competition of binding with soluble MOG documented their binding specificity. Such affinity purified Abs labeled myelin and glial cells in human CNS white matter as well as myelinated axons in gray matter. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG Abs, compared with 14.7% of patients in the 10- to 18-year age group. B cell autoimmunity to this myelin surface Ag is therefore most common in patients with a very early onset of MS.

Antibodies Produced by Clonally Expanded Plasma Cells in Multiple Sclerosis Cerebrospinal Fluid

Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B‐cell clonal expansion are well‐characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic.

The neuroimmunology of multiple sclerosis: possible roles of T and B lymphocytes in immunopathogenesis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system white matter. The association of the disease with MHC genes, the inflammatory white matter infiltrates, similarities with animal models, and the observation that MS can be treated with immunomodulatory and immunosuppressive therapies support the hypothesis that autoimmunity plays a major role in the disease pathology. Evidence supports activated CD4+ myelin-reactive T cells as major mediators of the disease. In addition, a renewed interest in the possible contribution of B cells to MS immunopathology has been sparked by nonhuman primate and MS pathological studies. This review focuses on the immunopathology of MS, outlining the hypothetical steps of tolerance breakdown and the molecules that play a role in the migration of autoreactive cells to the CNS. Particular focus is given to autoreactive T cells and cytokines as well as B cells and autoantibodies and their role in CNS pathogenesis in MS.

Antibodies from Inflamed Central Nervous System Tissue Recognize Myelin Oligodendrocyte Glycoprotein

Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) can induce demyelination and oligodendrocyte loss in models of multiple sclerosis (MS). Whether anti-MOG Abs play a similar role in patients with MS or inflammatory CNS diseases by epitope spreading is unclear. We have therefore examined whether autoantibodies that bind properly folded MOG protein are present in the CNS parenchyma of MS patients. IgG was purified from CNS tissue of 14 postmortem cases of MS and 8 control cases, including cases of encephalitis. Binding was assessed using two independent assays, a fluorescence-based solid-phase assay and a solution-phase RIA. MOG autoantibodies were identified in IgG purified from CNS tissue by solid-phase immunoassay in 7 of 14 cases with MS and 1 case of subacute sclerosing panencephalitis, but not in IgG from noninflamed control tissue. This finding was confirmed with a solution-phase RIA, which measures higher affinity autoantibodies. These data demonstrate that autoantibodies recognizing MOG are present in substantially higher concentrations in the CNS parenchyma compared with cerebrospinal fluid and serum in subjects with MS, indicating that local production/accumulation is an important aspect of autoantibody-mediated pathology in demyelinating CNS diseases. Moreover, chronic inflammatory CNS disease may induce autoantibodies by virtue of epitope spreading.

Plasma cells in muscle in inclusion body myositis and polymyositis

Background: Previous immunohistochemical studies of muscle from patients with inclusion body myositis and polymyositis found many more T cells than B cells, suggesting a role for intramuscular cell-mediated immune mechanisms rather than humoral mechanisms. Methods: Microarray studies were performed on muscle biopsy specimens from 40 patients with inclusion body myositis (IBM; n = 23), polymyositis (PM; n = 6), and without neuromuscular disease (n = 11). Reverse transcription PCR of selected immunoglobulin gene transcripts was performed on two patient samples. Qualitative immunohistochemical studies for B-cell lineage cell surface markers were performed on 28 muscle specimens and quantitative studies performed on a subset of 19 untreated patients with IBM or PM. CD138+ cells were isolated from muscle using laser capture microdissection, and immunoglobulin transcripts were PCR amplified to determine the presence or absence of immunoglobulin gene rearrangements unique to the B-cell lineage. Results: Immunoglobulin gene transcripts accounted for 59% in IBM and 33% in PM of the most stringently defined highest differentially expressed muscle transcripts compared with normal. Plasma cells, terminally differentiated B cells expressing CD138 but not CD19 or CD20, are present in IBM and PM muscle in numbers several times higher than B cells. Conclusions: There are differentiated B cells in the form of CD138+ plasma cells within the muscle of patients with inclusion body myositis and polymyositis. The principle of linked recognition of B-cell activation predicts several strategies for autoantigen discovery that could not otherwise be pursued through the study of the infiltrating T-cell population alone.

A Local Antigen-Driven Humoral Response Is Present in the Inflammatory Myopathies

The inflammatory myopathies are putative autoimmune disorders characterized by muscle weakness and the presence of intramuscular inflammatory infiltrates. Although inclusion body myositis and polymyositis have been characterized as cytotoxic CD8+ T cell-mediated diseases, we recently demonstrated high frequencies of CD138+ plasma cells in the inflamed muscle tissue of patients with these diseases. To gain a deeper understanding of the role these B cell family members play in the disease pathology, we examined the molecular characteristics of the H chain portion of the Ag receptor. Biopsies of muscle tissue were sectioned and tissue regions and individual cells were isolated through laser capture microdissection. Ig H chain gene transcripts isolated from the sections, regions, and cells were used to determine the variable region gene sequences. Analysis of these sequences revealed clear evidence of affinity maturation in that significant somatic mutation, isotype switching, receptor revision, codon insertion/deletion, and oligoclonal expansion had occurred within the B and plasma cell populations. Moreover, analysis of tissue regions isolated by laser capture microdissection revealed both clonal expansion and variation, suggesting that local B cell maturation occurs within muscle. In contrast, sequences from control muscle tissues and peripheral blood revealed none of these characteristics found in inflammatory myopathy muscle tissue. Collectively, these data demonstrate that Ag drives a B cell Ag-specific response in muscle in patients with dermatomyositis, inclusion body myositis, and polymyositis. These findings highlight the need for a revision of the current paradigm of exclusively T cell-mediated intramuscular Ag-specific autoimmunity in inclusion body myositis and polymyositis.

MOG cell-based assay detects non-MS patients with inflammatory neurologic disease

Objective: To optimize sensitivity and disease specificity of a myelin oligodendrocyte glycoprotein (MOG) antibody assay. Methods: Consecutive sera (n = 1,109) sent for aquaporin-4 (AQP4) antibody testing were screened for MOG antibodies (Abs) by cell-based assays using either full-length human MOG (FL-MOG) or the short-length form (SL-MOG). The Abs were initially detected by Alexa Fluor goat anti-human IgG (H + L) and subsequently by Alexa Fluor mouse antibodies to human IgG1. Results: When tested at 1:20 dilution, 40/1,109 sera were positive for AQP4-Abs, 21 for SL-MOG, and 180 for FL-MOG. Only one of the 40 AQP4-Ab–positive sera was positive for SL-MOG-Abs, but 10 (25%) were positive for FL-MOG-Abs (p = 0.0069). Of equal concern, 48% (42/88) of sera from controls (patients with epilepsy) were positive by FL-MOG assay. However, using an IgG1-specific secondary antibody, only 65/1,109 (5.8%) sera were positive on FL-MOG, and AQP4-Ab– positive and control sera were negative. IgM reactivity accounted for the remaining anti-human IgG (H + L) positivity toward FL-MOG. The clinical diagnoses were obtained in 33 FL-MOG–positive patients, blinded to the antibody data. IgG1-Abs to FL-MOG were associated with optic neuritis (n = 11), AQP4-seronegative neuromyelitis optica spectrum disorder (n = 4), and acute disseminated encephalomyelitis (n = 1). All 7 patients with probable multiple sclerosis (MS) were MOG-IgG1 negative. Conclusions: The limited disease specificity of FL-MOG-Abs identified using Alexa Fluor goat anti-human IgG (H + L) is due in part to detection of IgM-Abs. Use of the FL-MOG and restricting to IgG1-Abs substantially improves specificity for non-MS demyelinating diseases. Classification of evidence: This study provides Class II evidence that the presence of serum IgG1- MOG-Abs in AQP4-Ab–negative patients distinguishes non-MS CNS demyelinating disorders from MS (sensitivity 24%, 95% confidence interval [CI] 9%–45%; specificity 100%, 95% CI 88%–100%).