Short- and Long-Lived Autoantibody-Secreting Cells in Autoimmune Neurological Disorders

As B cells differentiate into antibody-secreting cells (ASCs), short-lived plasmablasts (SLPBs) are produced by a primary extrafollicular response, followed by the generation of memory B cells and long-lived plasma cells (LLPCs) in germinal centers (GCs). Generation of IgG4 antibodies is T helper type 2 (Th2) and IL-4, -13, and -10-driven and can occur parallel to IgE, in response to chronic stimulation by allergens and helminths. Although IgG4 antibodies are non-crosslinking and have limited ability to mobilize complement and cellular cytotoxicity, when self-tolerance is lost, they can disrupt ligand-receptor binding and cause a wide range of autoimmune disorders including neurological autoimmunity. In myasthenia gravis with predominantly IgG4 autoantibodies against muscle-specific kinase (MuSK), it has been observed that one-time CD20+ B cell depletion with rituximab commonly leads to long-term remission and a marked reduction in autoantibody titer, pointing to a short-lived nature of autoantibody-secreting cells. This is also observed in other predominantly IgG4 autoantibody-mediated neurological disorders, such as chronic inflammatory demyelinating polyneuropathy and autoimmune encephalitis with autoantibodies against the Ranvier paranode and juxtaparanode, respectively, and extends beyond neurological autoimmunity as well. Although IgG1 autoantibody-mediated neurological disorders can also respond well to rituximab induction therapy in combination with an autoantibody titer drop, remission tends to be less long-lasting and cases where titers are refractory tend to occur more often than in IgG4 autoimmunity. Moreover, presence of GC-like structures in the thymus of myasthenic patients with predominantly IgG1 autoantibodies against the acetylcholine receptor and in ovarian teratomas of autoimmune encephalitis patients with predominantly IgG1 autoantibodies against the N‐methyl‐d‐aspartate receptor (NMDAR) confers increased the ability to generate LLPCs. Here, we review available information on the short-and long-lived nature of ASCs in IgG1 and IgG4 autoantibody-mediated neurological disorders and highlight common mechanisms as well as differences, all of which can inform therapeutic strategies and personalized medical approaches.

Complement activity in myasthenia gravis 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. Accordingly, complement inhibitors are used as a therapeutic strategy, but the response can be heterogeneous even though AChR autoantibodies are present. The mechanisms underlying the variable response are not defined. Yet there is a need for further understanding so that responses can be better predicted. There is a broad spectrum of circulating complement activity levels activity among MG patients. It is not clear whether this activity associates with disease burden or the circulating levels of autoantibodies. We measured complement activity and investigated these associations in MG patients as a means to explore candidate biomarkers. Most study subjects had complement activity within the range defined by healthy controls and no association between this activity and disease burden or AChR autoantibody titer was observed. Assays measuring the complement activating properties of AChR autoantibodies are needed to identifying patients expected to respond to complement inhibitor-based treatments.

Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones.

Bortezomib Yields High Response Rates in Antibody-Mediated Autoimmune Hematological Diseases Refractory to Conventional Immunosuppression

Introduction Certain patients with antibody-mediated autoimmune disease exhibit poor responses to conventional immunosuppression. Proteasome inhibitors (PI) have been prospectively evaluated in humoral graft rejection, where rapid alloantibody depletion occurs in the context of combined immunosuppression. In addition to killing antibody-producing cells, PIs deplete autoreactive T-cells, suppress inflammatory NFkB signaling and modulate MHC class I antigen presentation. The durability of rituximab-based B-cell depletion may be abrogated by promotion of long-lived autoreactive (CD20 neg) plasma cell development. Therefore there is a strong rational for utilising PIs in antibody mediated autoimmune disease. Based on our initial experience re-purposing bortezomib (BTZ) to deplete ADAMTS-13 antibodies in thrombotic thrombocytopenic purpura (TTP) (Shortt et al NEJM 2013; 69: 90-2.), we have utilized off-label BTZ in cases of severe refractory antibody-mediated hematological disease via a compassionate access program. We report the first case-series of BTZ use in this broader patient group. Patients & Methods Outcome data were collected for all patients treated between 2012 and 2015 with off-label BTZ salvage for autoantibody-mediated hematological disease across a combined health-care network in the Australian state of Victoria (catchment area >1.5 million patients; 3 major academic centers). Eligible patients were refractory to standard of care and at risk of severe morbidity/mortality from underlying disease or immunosuppression. All patients demonstrated on-going autoantibody production despite rituximab-based B-cell depletion. We sought to correlate biological responses (e.g. reductions in autoantibody titer) with clinical efficacy (e.g. remission rates and capacity to wean concurrent immunosuppression) and to document BTZ toxicities. Compassionate BTZ access was subject to institutional drug and therapeutic committee review and Australian Therapeutic Goods Administration notification. Routine antiviral prophylaxis was administered. Results: Treatment episodes (n=8) included TTP (n=3), warm autoimmune hemolytic anemia (AIHA) (n=3), cold AIHA (n=1) and an acquired FVIII inhibitor (n=1) in 7 patients (median age 53 years, range 34-80; M/F 4:3). Patients had received a median of 3.5 prior lines of therapy (range: 2-6) and all were rituximab exposed. BTZ was predominantly administered at 1.3mg/m2; D1, 4, 8, 11 q21d, with subsequent dose modification to weekly where repeated cycles were required. Initial IV therapy was preferred in TTP patients, to maximize exposure between exchanges; where recurrent cycles were administered all patients transitioned to subcutaneous administration to avoid neurotoxicity. With a median follow-up of 10 months (censored June 2015; range: 4-35 months), the overall response rate was 75%, including 50% durable complete remissions and 25% partial remission with reductions in transfusion requirements and baseline immunosuppression. Biological responses correlated with reductions in autoantibody titer. Most patients demonstrated an immediate response within 1 cycle (median cycles delivered 1.5; range: 1-6). BTZ was generally well tolerated with grade 4 hepatotoxicity observed in one patient in the context of high alcohol intake. No infective complications or neuropathy were observed. Table 1.CaseAgeM/FDiseasePrior Lines of TreatmentCyclesResponseDuration of response170MAIHA (warm)31NR02a53FTTP41CR7m2b54FTTP21CR25m364FAIHA (cold)56PR5m449FTTP21CR32m534MHemophilia A34CR4m647MAIHA (warm)52PR2m780MAIHA (warm)62NR0NR - no response; CR - complete response; PR - partial response (reduction in immunosuppression or disease activity) Conclusions: BTZ rapidly downregulates autoantibodies, correlating with a high response rate in relapsed/refractory autoimmune hematological disease with an acceptable/expected toxicity profile. This case series, taken together with emerging case reports of efficacy provide 'proof of concept' for the utility of PI in antibody mediated autoimmune disease. A prospective clinical trial protocol is in development. Disclosures Off Label Use: The drug to be discussed is bortezomib and its use in managing refractory autoimmune haematological disorders (off label use). Catalano:Celgene, Roche, Gilead: Honoraria. Opat:Janssen Pharmaceuticals: Other: Provision of subsidized medications only. Shortt:Janssen Pharmaceuticals: Other: Provision of subsidized medications only.

Monitoring ADAMTS13 Autoantibody Titer Using a Novel Quantitative Radioimmunoprecipitation Assay In Patients With Acquired Thrombotic Thrombocytopenic Purpura

Background ADAMTS13-binding immunoglobulin G (IgG)-type autoantibodies are present in patients with acquired thrombotic thrombocytopenic purpura (TTP), thereby causing severe deficiency of plasma ADAMTS13 activity. Some specific autoantibodies directly inhibit the enzymatic activity by interfering with the access of its substrate, von Willebrand factor (VWF), particularly in the spacer domain, although others bind to almost all of the domains in the molecule, possibly leading to the acceleration of the clearance from blood stream. Not only the inhibitor titer, but the importance of ADAMTS13 autoantibody titer was highlighted by many previous clinical studies, associating with the prognosis such as recurrence rate. Objective We targeted to establish a novel high-sensitive assay to measure ADAMTS13-binding IgG autoantibody titer using three kinds of radioisotope-labeled antigens, ADAMTS13 whole molecule, MDTCS and T2-8/CUB, and aimed to analyze the association between the autoantibody titer and the clinical characteristics of TTP patients. Materials and Methods Human Cell-Free Protein Expression System (Takara #3281, Shiga, Japan) was used to synthesize radioisotope-labeled antigens. ADAMTS13 cDNA corresponding to whole molecule (A13), metalloprotease to spacer domains (MDTCS) and TSP1-2 to CUB2 domains (T2-8/CUB) were cloned into an expression vector pT7-IRES and mixed with Cell Lysate containing T7 RNA polymerase, methionine-free amino acids, ATP, translation enhancement factor and 35S-methionine. The correct synthesis and molecular size of the radiolabeled antigens were checked with SDS-PAGE. To assess the utility as a quantitative assay, each of the antigens was mixed with mouse anti-ADAMTS13 monoclonal antibodies, whose epitopes were determined in our previous study (Thromb Res. 2012; 130(3):e79-83), and the immune complex was precipitated with protein G beads, washed and measured in a liquid scintillation counter. Plasma samples from acquired TTP patients were tested to quantify the autoantibody titers using radiolabeled A13, MDTCS and T2-8/CUB antigens, respectively. As a control, plasma samples from healthy subjects with no histories of autoimmune disease were also tested. Results Each of the radiolabeled antigens was detected as a single band at the correct molecular weight size and successfully immnoprecipitated with several mouse anti-ADAMTS13 monoclonal antibodies, indicating the intact molecular conformation of the synthesized proteins using the cell-free protein synthesis system. Moreover, the appropriate dose-dependent escalation curves in accordance with the addition of the monoclonal antibodies were observed, thereby confirming the utility of the assay as a quantitative analysis. We tested TTP patient plasma at onset (n=5) and were able to detect ADAMTS13 autoantibody titers with each of the radiolabeled A13, MDTCS and T2-8/CUB antigens. We next applied this assay for monitoring ADAMTS13 autoantibody titer in a clinical course of TTP patient. The patient developed the first episode of TTP at the age of 2 month and treated with steroid pulse and plasma exchange therapy for six consecutive days. Remission was once achieved but 6 months later from the onset, the second episode of TTP occurred and the patient was treated with 11 plasma exchange and rituximab at the dose of 375 mg/m2 once a week for 4 weeks. Plasma samples at onset, after the first 6 consecutive plasma exchange and after rituximab administration were examined about autoantibody titer using this assay. Interestingly, the titers remained high even after the plasma exchange but declined clearly after the rituximab treatment, whereas no reduction of total IgG level was observed. These findings suggest that the autoantibody titration using this assay might be useful to assess the effect of treatment and associate with the prognosis related to the recurrence. Conclusion We developed a novel quantitative radioimmunoprecipitation assay to measure ADAMTS13 autoantibody titer related to three antigens, ADAMTS13 whole molecule, MDTCS and TSP2-8/CUB. This assay may serve not only as a diagnostic test but as a monitoring index to evaluate the prognosis of TTP. Disclosures: No relevant conflicts of interest to declare.

Low HEMA Conjugation Induces High Autoantibody Titer in Mice

2-hydroxyethylmethacrylate (HEMA) is a known causal agent of hypersensitivity to resin composites. We have reported that immunization with HEMA conjugated to mouse serum albumin (MSA) induces an autoantibody response in mice. In this study, we investigated both the activity and the avidity of autoantibodies induced by immunization with various HEMA conjugations to MSA. Female Balb/c mice were given MSA carrying 3, 7, 15, or 22 HEMA molecules. Antigen-specific IgG and IgE antibodies were determined by ELISA, and average antibody avidity by thiocyanate dissociation. Immunization with MSA carrying the lowest number of HEMA molecules induced a significantly higher IgG and IgE anti-MSA autoantibody response, with significantly higher IgG antibody avidity, than did the more heavily conjugated preparations. The results suggest that the lower the degree of HEMA conjugation to self-protein, the higher the risk for autoantibody production to the carrier protein. These findings suggest a mechanism of potential relevance in humans.