A Noncanonical CD56dimCD16dim/- NK Cell Subset Indicative of Prior Cytotoxic Activity Is Elevated in Patients with Autoantibody-Mediated Neurologic Diseases.

Neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein Ab disease, and autoimmune myasthenia gravis (MG) are autoantibody-mediated neurologic conditions where autoantibodies can induce Ab-dependent cellular cytotoxicity (ADCC), a NK cell-mediated effector function. However, whether ADCC is a pathogenic mechanism in patients with these conditions has not been confirmed. We sought to characterize circulatory NK cells using functional assays, phenotyping, and transcriptomics to elucidate their role in pathology. NK cells from NMOSD patients and MG patients with elevated disease burden exhibited reduced ADCC and CD56dimCD16hi NK cells, along with an elevated frequency of CD56dimCD16dim/- NK cells. We determined that ADCC induces a similar phenotypic shift in vitro. Bulk RNA sequencing distinguished the CD56dimCD16dim/- population from the canonical CD56dimCD16hi cytotoxic and CD56hiCD16- immunomodulatory subsets, as well as CD56hiCD16+ NK cells. Multiparameter immunophenotyping of NK cell markers, functional proteins, and receptors similarly showed that the CD56dimCD16dim/- subset exhibits a unique profile while still maintaining expression of characteristic NK markers CD56, CD94, and NKp44. Notably, expression of perforin and granzyme is reduced in comparison with CD56dimCD16hi NK cells. Moreover, they exhibit elevated trogocytosis capability, HLA-DR expression, and many chemokine receptors, including CCR7. In contrast with NMOSD and MG, myelin oligodendrocyte glycoprotein Ab disease NK cells did not exhibit functional, phenotypic, or transcriptomic perturbations. In summary, CD56dimCD16dim/- NK cells are a distinct peripheral blood immune cell population in humans elevated upon prior cytotoxic activity by the CD56dimCD16hi NK cell subset. The elevation of this subset in NMOSD and MG patients suggests prior ADCC activity.

From drab to Fab; PLP1's fit is redressed for MS.

Curated expression of proteolipid protein 1 (PLP1) is essential for multiple sclerosis-derived autoantibody recognition.

Remission of severe myasthenia gravis after autologous stem cell transplantation.

OBJECTIVE: Myasthenia gravis (MG) is an autoantibody-mediated neuromuscular junction disorder involving the acetylcholine receptors on the motor endplate. The safety and response to high-dose chemotherapy (HDIT) and autologous hematopoietic cell transplantation (HCT) were assessed in a patient with severe refractory MG. METHODS: As part of a pilot study of HDIT/HCT for patients with treatment-resistant autoimmune neurological disorders, a patient with severe refractory MG underwent treatment. After mobilization of hematopoietic stem cells with rituximab, prednisone, and G-CSF, the patient had HDIT consisting of carmustine, etoposide, cytarabine, melphalan, and rabbit antithymocyte globulin, followed by autologous HCT. The effect of treatment on the autoantibody to the acetylcholine receptor (AChR) was assessed. RESULTS: The patient had been diagnosed with AChR antibody-positive MG 14 years before HDIT/HCT and had failed thymectomy, therapeutic plasma exchange, and multiple immunomodulatory agents. The Myasthenia Gravis Foundation of America (MGFA) clinical classification was IVb before HDIT/HCT. She tolerated HDIT/HCT well and started to improve clinically within days of treatment. At both 1 and 2 years after HDIT/HCT, patients remained symptom-free. After HDIT/HCT, AChR-binding autoantibodies persisted, and the relative frequency of immune cell subtypes shifted. INTERPRETATION: HDIT/HCT induced a complete response of disease activity in a patient with severe refractory MG. This response may suggest that a cell-mediated etiology may be a significant contributing factor in refractory MG cases. A phase 2 clinical trial is warranted to establish if HDIT/HCT can be an effective therapy for severe refractory MG and to gain a further understanding of disease pathogenesis.

Toddler's T cells are taught in muco-school.

Tissue-resident memory T cells accumulate in mucosal sites during infancy and then mature through childhood.

Individual myasthenia gravis autoantibody clones can efficiently mediate multiple mechanisms of pathology.

Serum autoantibodies targeting the nicotinic acetylcholine receptor (AChR) in patients with autoimmune myasthenia gravis (MG) can mediate pathology via three distinct molecular mechanisms: complement activation, receptor blockade, and antigenic modulation. However, it is unclear whether multi-pathogenicity is mediated by individual or multiple autoantibody clones. Using an unbiased B cell culture screening approach, we generated a library of 11 human-derived AChR-specific recombinant monoclonal autoantibodies (mAb) and assessed their binding properties and pathogenic profiles using specialized cell-based assays. Five mAbs activated complement, three blocked α-bungarotoxin binding to the receptor, and seven induced antigenic modulation. Furthermore, two clonally related mAbs derived from one patient were each highly efficient at more than one of these mechanisms, demonstrating that pathogenic mechanisms are not mutually exclusive at the monoclonal level. Using novel Jurkat cell lines that individually express each monomeric AChR subunit (α2ßδε), these two mAbs with multi-pathogenic capacity were determined to exclusively bind the α-subunit of AChR, demonstrating an association between mAb specificity and pathogenic capacity. These findings provide new insight into the immunopathology of MG, demonstrating that single autoreactive clones can efficiently mediate multiple modes of pathology. Current therapeutic approaches targeting only one autoantibody-mediated pathogenic mechanism may be evaded by autoantibodies with multifaceted capacity.

The Plasma Cell Infiltrate Populating the Muscle Tissue of Patients with Inclusion Body Myositis Features Distinct B Cell Receptor Repertoire Properties.

Inclusion body myositis (IBM) is an autoimmune and degenerative disorder of skeletal muscle. The B cell infiltrates in IBM muscle tissue are predominantly fully differentiated Ab-secreting plasma cells, with scarce naive or memory B cells. The role of this infiltrate in the disease pathology is not well understood. To better define the humoral response in IBM, we used adaptive immune receptor repertoire sequencing, of human-derived specimens, to generate large BCR repertoire libraries from IBM muscle biopsies and compared them to those generated from dermatomyositis, polymyositis, and circulating CD27+ memory B cells, derived from healthy controls and Ab-secreting cells collected following vaccination. The repertoire properties of the IBM infiltrate included the following: clones that equaled or exceeded the highly clonal vaccine-associated Ab-secreting cell repertoire in size; reduced somatic mutation selection pressure in the CDRs and framework regions; and usage of class-switched IgG and IgA isotypes, with a minor population of IgM-expressing cells. The IBM IgM-expressing population revealed unique features, including an elevated somatic mutation frequency and distinct CDR3 physicochemical properties. These findings demonstrate that some of IBM muscle BCR repertoire characteristics are distinct from dermatomyositis and polymyositis and circulating Ag-experienced subsets, suggesting that it may form through selection by disease-specific Ags.

MOGAD patient autoantibodies induce complement, phagocytosis, and cellular cytotoxicity.

Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an inflammatory demyelinating CNS condition characterized by the presence of MOG autoantibodies. We sought to investigate whether human MOG autoantibodies are capable of mediating damage to MOG-expressing cells through multiple mechanisms. We developed high-throughput assays to measure complement activity (CA), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC) of live MOG-expressing cells. MOGAD patient sera effectively mediate all of these effector functions. Our collective analyses reveal that (a) cytotoxicity is not incumbent on MOG autoantibody quantity alone; (b) engagement of effector functions by MOGAD patient serum is bimodal, with some sera exhibiting cytotoxic capacity while others did not; (c) the magnitude of CDC and ADCP is elevated closer to relapse, while MOG-IgG binding is not; and (d) all IgG subclasses can damage MOG-expressing cells. Histopathology from a representative MOGAD case revealed congruence between lesion histology and serum CDC and ADCP, and we identified NK cells, mediators of ADCC, in the cerebrospinal fluid of relapsing patients with MOGAD. Thus, MOGAD-derived autoantibodies are cytotoxic to MOG-expressing cells through multiple mechanisms, and assays quantifying CDC and ADCP may prove to be effective tools for predicting risk of future relapses.

SAkuraBONSAI: Protocol design of a novel, prospective study to explore clinical, imaging, and biomarker outcomes in patients with AQP4-IgG-seropositive neuromyelitis optica spectrum disorder receiving open-label satralizumab.

Background: Neuromyelitis optica spectrum disorder (NMOSD) is a rare, autoimmune disease of the central nervous system that produces acute, unpredictable relapses causing cumulative neurological disability. Satralizumab, a humanized, monoclonal recycling antibody that targets the interleukin-6 receptor, reduced NMOSD relapse risk vs. placebo in two Phase 3 trials: SAkuraSky (satralizumab ± immunosuppressive therapy; NCT02028884) and SAkuraStar (satralizumab monotherapy; NCT02073279). Satralizumab is approved to treat aquaporin-4 IgG-seropositive (AQP4-IgG+) NMOSD. SAkuraBONSAI (NCT05269667) will explore fluid and imaging biomarkers to better understand the mechanism of action of satralizumab and the neuronal and immunological changes following treatment in AQP4-IgG+ NMOSD. Objectives: SAkuraBONSAI will evaluate clinical disease activity measures, patient-reported outcomes (PROs), pharmacokinetics, and safety of satralizumab in AQP4-IgG+ NMOSD. Correlations between imaging markers (magnetic resonance imaging [MRI] and optical coherence tomography [OCT]) and blood and cerebrospinal fluid (CSF) biomarkers will be investigated. Study design: SAkuraBONSAI is a prospective, open-label, multicenter, international, Phase 4 study that will enroll approximately 100 adults (18-74 years) with AQP4-IgG+ NMOSD. This study includes two patient cohorts: newly diagnosed, treatment-naïve patients (Cohort 1; n = 60); and inadequate responders to recent (<6 months) rituximab infusion (Cohort 2; n = 40). Satralizumab monotherapy (120 mg) will be administered subcutaneously at Weeks 0, 2, 4, and Q4W thereafter for a total of 92 weeks. Endpoints: Disease activity related to relapses (proportion relapse-free, annualized relapse rate, time to relapse, and relapse severity), disability progression (Expanded Disability Status Scale), cognition (Symbol Digit Modalities Test), and ophthalmological changes (visual acuity; National Eye Institute Visual Function Questionnaire-25) will all be assessed. Peri-papillary retinal nerve fiber layer and ganglion cell complex thickness will be monitored using advanced OCT (retinal nerve fiber layer and ganglion cell plus inner plexiform layer thickness). Lesion activity and atrophy will be monitored by MRI. Pharmacokinetics, PROs, and blood and CSF mechanistic biomarkers will be assessed regularly. Safety outcomes include the incidence and severity of adverse events. Conclusions: SAkuraBONSAI will incorporate comprehensive imaging, fluid biomarker, and clinical assessments in patients with AQP4-IgG+ NMOSD. SAkuraBONSAI will provide new insights into the mechanism of action of satralizumab in NMOSD, while offering the opportunity to identify clinically relevant neurological, immunological, and imaging markers.

Plasmablasts from the past: Nostalgic B cells can't let go.

An approach for identifying antibodies derived from distinct B cell populations demonstrates how secondary immunization responses are dominated by mature B cells generated during primary responses.

Clinicoserological insights into patients with immune checkpoint inhibitor-induced myasthenia gravis.

To compare the immunopathology of immune checkpoint inhibitor-induced myasthenia gravis (ICI-MG) and idiopathic MG, we profiled the respective AChR autoantibody pathogenic properties. Of three ICI-MG patients with AChR autoantibodies, only one showed complement activation and modulation/blocking potency, resembling idiopathic MG. In contrast, AChR autoantibody-mediated effector functions were not detected in the other two patients, questioning the role of their AChR autoantibodies as key mediators of pathology. The contrasting properties of AChR autoantibodies in these cases challenge the accuracy of serological testing in establishing definite ICI-MG diagnoses and underscore the importance of a thorough clinical assessment when evaluating ICI-related adverse events.

Precision targeting of autoantigen-specific B cells in muscle-specific tyrosine kinase myasthenia gravis with chimeric autoantibody receptor T cells.

Muscle-specific tyrosine kinase myasthenia gravis (MuSK MG) is an autoimmune disease that causes life-threatening muscle weakness due to anti-MuSK autoantibodies that disrupt neuromuscular junction signaling. To avoid chronic immunosuppression from current therapies, we engineered T cells to express a MuSK chimeric autoantibody receptor with CD137-CD3ζ signaling domains (MuSK-CAART) for precision targeting of B cells expressing anti-MuSK autoantibodies. MuSK-CAART demonstrated similar efficacy as anti-CD19 chimeric antigen receptor T cells for depletion of anti-MuSK B cells and retained cytolytic activity in the presence of soluble anti-MuSK antibodies. In an experimental autoimmune MG mouse model, MuSK-CAART reduced anti-MuSK IgG without decreasing B cells or total IgG levels, reflecting MuSK-specific B cell depletion. Specific off-target interactions of MuSK-CAART were not identified in vivo, in primary human cell screens or by high-throughput human membrane proteome array. These data contributed to an investigational new drug application and phase 1 clinical study design for MuSK-CAART for the treatment of MuSK autoantibody-positive MG.

Unveiling the autoreactome: Proteome-wide immunological fingerprints reveal the promise of plasma cell depleting therapy.

The prevalence and burden of autoimmune and autoantibody mediated disease is increasing worldwide, yet most disease etiologies remain unclear. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leverage advances in programmable-phage immunoprecipitation (PhIP-Seq) methodology to explore the modulation, or lack thereof, of proteome-wide autoantibody profiles in both health and disease. We demonstrate that each individual, regardless of disease state, possesses a distinct set of autoreactivities constituting a unique immunological fingerprint, or "autoreactome", that is remarkably stable over years. In addition to uncovering important new biology, the autoreactome can be used to better evaluate the relative effectiveness of various therapies in altering autoantibody repertoires. We find that therapies targeting B-Cell Maturation Antigen (BCMA) profoundly alter an individual's autoreactome, while anti-CD19 and CD-20 therapies have minimal effects, strongly suggesting a rationale for BCMA or other plasma cell targeted therapies in autoantibody mediated diseases.

Reemergence of pathogenic, autoantibody-producing B cell clones in myasthenia gravis following B cell depletion therapy.

Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (<10%) with MG, have autoantibodies targeting muscle-specific tyrosine kinase (MuSK). MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we studied autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an acetylcholine receptor (AChR) clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These findings demonstrate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. Overall, this study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction.

EBVerified TCRs and multiple sclerosis.

Patients with multiple sclerosis display broad EBV-specific TCR repertoires driven by an enduring anti-EBV immune response.

Novel pathophysiological insights in autoimmune myasthenia gravis.

PURPOSE OF REVIEW: This review summarizes recent insights into the immunopathogenesis of autoimmune myasthenia gravis (MG). Mechanistic understanding is presented according to MG disease subtypes and by leveraging the knowledge gained through the use of immunomodulating biological therapeutics. RECENT FINDINGS: The past two years of research on MG have led to a more accurate definition of the mechanisms through which muscle-specific tyrosine kinase (MuSK) autoantibodies induce pathology. Novel insights have also emerged from the collection of stronger evidence on the pathogenic capacity of low-density lipoprotein receptor-related protein 4 autoantibodies. Clinical observations have revealed a new MG phenotype triggered by cancer immunotherapy, but the underlying immunobiology remains undetermined. From a therapeutic perspective, MG patients can now benefit from a wider spectrum of treatment options. Such therapies have uncovered profound differences in clinical responses between and within the acetylcholine receptor and MuSK MG subtypes. Diverse mechanisms of immunopathology between the two subtypes, as well as qualitative nuances in the autoantibody repertoire of each patient, likely underpin the variability in therapeutic outcomes. Although predictive biomarkers of clinical response are lacking, these observations have ignited the development of assays that might assist clinicians in the choice of specific therapeutic strategies. SUMMARY: Recent advances in the understanding of autoantibody functionalities are bringing neuroimmunologists closer to a more detailed appreciation of the mechanisms that govern MG pathology. Future investigations on the immunological heterogeneity among MG patients will be key to developing effective, individually tailored therapies.

Identity thieves: T cells steal CD20 from B cells but mark themselves for certain death.

T cells can acquire CD20 from B cells via trogocytosis, then contribute to autoimmune neurological disease while becoming targets for therapeutically effective B cell depletion.

The clinical need for clustered AChR cell-based assay testing of seronegative MG.

Trial eligibility in myasthenia gravis (MG) remains largely dependent on a positive autoantibody serostatus. This significantly hinders seronegative MG (SNMG) patients from receiving potentially beneficial new treatments. In a subset of SNMG patients, acetylcholine receptor (AChR) autoantibodies are detectable by a clustered AChR cell-based assay (CBA). Of 99 SNMG patients from two academic U.S. centers, 18 (18.2%) tested positive by this assay. Autoantibody positivity was further validated in 17/18 patients. In a complementary experiment, circulating AChR-specific B cells were identified in a CBA-positive SNMG patient. These findings corroborate the clinical need for clustered AChR CBA testing when evaluating SNMG patients.

Heterogeneity of Acetylcholine Receptor Autoantibody-Mediated Complement Activity in Patients With Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Autoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. METHODS: An HEK293T cell line-modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)-was used to measure AChR autoantibody-mediated MAC formation through flow cytometry. RESULTS: Serum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58-1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10-90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, p = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset. DISCUSSION: A novel assay for evaluating AChR autoantibody-mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.

Myasthenia gravis complement activity is independent of autoantibody titer and disease severity.

Acetylcholine receptor (AChR) autoantibodies, found in patients with autoimmune myasthenia gravis (MG), can directly contribute to disease pathology through activation of the classical complement pathway. Activation of the complement pathway in autoimmune diseases can lead to a secondary complement deficiency resulting in reduced complement activity, due to consumption, during episodes of disease activity. It is not clear whether complement activity in MG patients associates with measurements of disease activity or the titer of circulating pathogenic AChR autoantibodies. To explore such associations, as a means to identify a candidate biomarker, we measured complement activity in AChR MG samples (N = 51) using a CH50 hemolysis assay, then tested associations between these values and both clinical status and AChR autoantibody titer. The majority of the study subjects (88.2%) had complement activity within the range defined by healthy controls, while six patients (11.8%) showed reduced activity. No significant association between complement activity and disease status or AChR autoantibody titer was observed.

Reliability of patient self-reports to clinician-assigned functional scores of inclusion body myositis.

BACKGROUND: Sporadic inclusion body myositis (IBM) is a debilitating disease which leads to impaired ambulation and loss of hand function. Yale IBM Registry (IBMR) was launched in November 2016 to address the knowledge gap in IBM natural history data. The registry interface provides an IBM personalized index calculator (IBM-PIC) based on the IBM-functional rating scale (IBM-FRS). While the calculator is based on the IBM-FRS, it has not been directly compared to the IBM-FRS score. Therefore, in this study, we compared the patient-reported IBM-PIC score from this calculator with the physician-obtained IBM-FRS score. METHOD: IBM-FRS was administered over the phone within two weeks of their most recent IBM-PIC entry in the IBMR to 35 participants. To compare the agreement between IBM-FRS and IBM-PIC scores, Interrater Correlation Coefficient (ICC) analysis was performed. For individual questions, Fleiss Kappa statistics was used. RESULTS: Thirty-five active IBM-PIC users participated. Eighty percent of the participants were men, and 91% were White Caucasians. The reported IBM-FRS score of this group was 23.5 ± 7.4 (range 1-38). The Interrater Correlation Coefficient (ICC) between the physician-administered IBM-FRS score and the IBM-PIC was 0.98 (0.96-0.99). There was moderate to substantial agreement on all the questions on IBM-FRS except for handwriting and fine motor skills. DISCUSSION: IBM-PIC is a reliable indicator of the IBM-FRS score obtained by the physician. It is anticipated that this online platform will be a valuable tool for assessing IBM severity and monitoring disease progression remotely both in clinical practice and research studies.