Autoantibodies Produced at the Site of Tissue Damage Provide Evidence of Humoral Autoimmunity in Inclusion Body Myositis

Inclusion body myositis (IBM) belongs to a group of muscle diseases known as the inflammatory myopathies. The presence of antibody-secreting plasma cells in IBM muscle implicates the humoral immune response in this disease. However, whether the humoral immune response actively contributes to IBM pathology has not been established. We sought to investigate whether the humoral immune response in IBM both in the periphery and at the site of tissue damage was directed towards self-antigens. Peripheral autoantibodies present in IBM serum but not control serum recognized self-antigens in both muscle tissue and human-derived cell lines. To study the humoral immune response at the site of tissue damage in IBM patients, we isolated single plasma cells directly from IBM-derived muscle tissue sections and from these cells, reconstructed a series of recombinant immunoglobulins (rIgG). These rIgG, each representing a single muscle-associated plasma cell, were examined for reactivity to self-antigens. Both, flow cytometry and immunoblotting revealed that these rIgG recognized antigens expressed by cell lines and in muscle tissue homogenates. Using a mass spectrometry-based approach, Desmin, a major intermediate filament protein, expressed abundantly in muscle tissue, was identified as the target of one IBM muscle-derived rIgG. Collectively, these data support the view that IBM includes a humoral immune response in both the periphery and at the site of tissue damage that is directed towards self-antigens

Response of patients with refractory myasthenia gravis to rituximab: a retrospective study

Introduction: Myasthenia gravis, an autoimmune disorder of neuromuscular transmission, is treated by an array of immunomodulating therapies. A variable response is observed with certain patients being medically refractory. Methods: We report the results of 14 refractory generalized myasthenia gravis patients (6 AChR+; 8 MuSK+) treated with rituximab. Results: Sustained clinical improvement was observed in all patients as well as a reduction of conventional immunotherapies. Prednisone dose decreased a mean of 65.1%, 85.7%, and 93.8% after cycle 1, 2, and 3 of rituximab therapy, respectively. A statistically significant reduction in plasma exchange sessions was seen after cycle 1 with all patients being off of plasma exchange after cycle 3. Acetylcholine receptor antibody titers decreased a mean of 52.1% (p = 0.0046) post-cycle 2. Conclusion: Our results support the hypothesis that rituximab is beneficial and well tolerated in managing refractory myasthenia gravis and nearly doubles published cases. We propose that B-cell-directed therapies may become an attractive option and suggest pursuit of a prospective trial

Durability of the Rituximab Response in Acetylcholine Receptor Autoantibody-Positive Myasthenia Gravis

IMPORTANCE: Myasthenia gravis (MG), an autoimmune disorder of neuromuscular transmission, is treated by an array of immunotherapeutics, many of which are nonspecific. Even with current therapies, a subset of patients has medically refractory MG. The benefits of B-cell-targeted therapy with rituximab have been observed in MG; however, the duration of these benefits after treatment is unclear. OBJECTIVE: To evaluate the durability of response to rituximab in the treatment of acetylcholine receptor autoantibody-positive (AChR+) generalized MG. DESIGN, SETTING AND PARTICIPANTS: This retrospective case series study included 16 patients with AChR+ MG referred to an MG clinic from January 1, 2007, to December 31, 2015. The patients were treated with rituximab and followed up for 18 to 84 months after treatment. MAIN OUTCOMES AND MEASURES: Assessment of long-term clinical response, durability of response and/or relapse rate, AChR autoantibody levels, adverse effects, and inflammatory markers. RESULTS: In the 16 patients (6 men and 10 women; median age, 42 [range, 18-69] years), clinical improvement was observed in parallel with complete withdrawal or reduction of other immunotherapies, with all patients achieving complete stable remission, pharmacologic remission, or minimal manifestations based on the Myasthenia Gravis Foundation of America postintervention status criteria. Nine patients (56%) had a relapse during a mean follow-up of 36 (range, 24-47) months. Seven patients (44%) remained relapse free with a mean follow-up of 47 (range, 18-81) months since the last rituximab treatment. All values were normalized to a pretreatment anti-AChR antibody level of 100% and the mean levels after each rituximab cycle were calculated. A 33% decrease was seen after cycle 1 of rituximab treatment (100% vs 67%; P = .004); 20% after cycle 2 (compared with cycle 1) (67% vs 47%; P = .008); and 17% after cycle 3 (compared with cycle 2) (47% vs 30%; P = .02). However, the serum cytokine levels measured were found to be unchanged. CONCLUSIONS AND RELEVANCE: Rituximab therapy appears to be an effective option in patients with refractory AChR+ MG, who were observed to have a durable response after treatment. Identification of markers of disease relapse and sustained remission are critical next steps in the development of pathophysiology-relevant, evidence-based practice parameters for rituximab in the treatment of MG

Immunoassays evaluating recombinant IgG binding to Desmin.

<p>Independent immunoassays were used to demonstrate that recombinant immunoglobulin derived from an IBM muscle-associated plasma cell (rIgG-5) binds to human Desmin. (A) Human muscle homogenates were resolved by SDS-PAGE then transferred to membranes that were probed with IBM-derived recombinant rIgG-5 (lane 1) or a commercial anti-Desmin monoclonal antibody (lane 2). (B) Purified human Desmin was resolved by SDS-PAGE then transferred to membranes that were probed with IBM-derived rIgG-5 (lane 1) or rIgG-D3a (lane 2), which was derived from an immature antigen-inexperienced peripheral human B cell. (C) ELISA assays demonstrating IBM-derived immunoglobulin (rIgG-5) binding to the human muscle protein Desmin. ELISA plates were coated with either Desmin (left panel) or Vimentin (right panel) then probed with IBM rIgG-5 and a rIgG (D3a) derived from a single B cell from a normal healthy donor. Monoclonal antibodies (mAb) specific for each antigen were used as positive controls.</p

Cell-based assays to evaluate muscle-derived recombinant IgG autoreactivity.

<p>Recombinant IgG was prepared from single muscle tissue-associated plasma cells. A flow cytometry-based assay was then used to evaluate the binding of this rIgG to human muscle cell lines CCL-136, CRL-1598 and the human CNS-derived oligodendrocyte cell line, HOG. Representative positive staining shown in the histograms is indicated by an increase in median fluorescence intensity (MFI) of the IBM-derived rIgG (open curves) in comparison to staining with the secondary antibody alone (anti-human IgG-Alexa Fluor 647). A humanized monoclonal antibody, 8-18C5, specific for myelin oligodendrocyte glycoprotein served as negative control. The evaluation of extracellular binding revealed a modest increase in MFI for rIgG-5 (open curves) and rIgG-6 (open curves) with respect to the secondary antibody for both muscle-derived cell lines, but not the CNS-derived cell line. Both rIgG-5 and rIgG-6 demonstrate detectable binding to all three permeabilized cell lines.</p

Cell-based assays to evaluate serum autoreactivity.

<p>A flow cytometry-based assay was used to evaluate IBM serum-derived IgG binding to human muscle-derived cell lines CCL-136, CRL-1598 and the human CNS-derived oligodendrocyte cell line, HOG. (A) Positive staining in the representative histograms is indicated by an increase in median fluorescence intensity (MFI) of an IBM-derived IgG (open curves) in comparison to a representative healthy control-derived IgG (closed curves). Specimen IBM001 binds to permeabilized muscle (CCL-136, CRL-1598) and non-muscle (HOG) cells but does not bind to cell surface components (extracellular staining). IBM003 binds to cell surface components of CRL-1598 while IBM005 binds to both CCL-136 and CRL-1598 cell lines. Both, IBM003 and IBM005 bind to permeabilized muscle cells (CCL-136 and CRL-1598). IBM003 binds to both intra- and extra-cellular components of the HOG line while IBM005 does not bind to either intracellular or extracellular components on the HOG cell line. The MFI values for all of the IBM serum-derived IgG specimens and controls are shown graphically. Assays performed with each of the three cell lines to measure binding to extracellular (B) and permeabilized cell lines (C) were collected for two different populations (healthy subjects and IBM patients). Each data point represents a single subject. The threshold line, above which specimens are considered positive, represents the mean +2 standard deviations of the normal healthy control cohort. A subset of IgG derived from IBM bind to self-antigens expressed within and on the surface of the cell lines tested.</p

Autoantigen isolation and identification strategy.

<p>Recombinant IgG derived from an IBM muscle plasma cell was used to isolate an unidentified target antigen. Muscle tissue homogenates (25 µg) were resolved by SDS-PAGE, then stained with coomassie brilliant blue or transferred to nitrocellulose. Molecular weight markers (kDa) are indicated on the left of all panels. An immunoblot (A) of muscle tissue homogenate probed with rIgG derived from a single IBM-tissue associated plasma cell (rIgG-5) demonstrated binding to a candidate antigen (lane 1). Binding was not present with the rIgG-2 (lane 2), derived from a different muscle specimen or with control antibodies, D3a (lane 3) and 18/2 (lane 4) that were derived from a healthy donor and from a patient with lupus respectively. A second immunoblot (bottom) probed with anti-HSP70 served as a loading control. (B) Two separate human skeletal muscle homogenates prepared using either stringent (lane 1) or mild protein extraction conditions (lane 2) were resolved by SDS-PAGE gel, and then transferred to nitrocellulose. Immunoblotting with rIgG-5 (lane 1), demonstrated the target antigen was present when the homogenate was prepared with the stringent extraction, but not with the mild conditions (lane 2). An immunoblot (bottom) with HSP70 served as a loading control. (C) To isolate the candidate antigen, the two homogenates were separated by SDS-PAGE<b>,</b> then bands with the approximate molecular weight of the candidate antigen were excised from the gel. Lane 1, muscle homogenized with the stringent extraction procedure; Lane 2, molecular weight markers; Lane 3, muscle homogenized with the mild extraction procedure. These excised proteins were digested with trypsin and the resulting peptides separated by reverse-phase chromatography, then applied to a mass spectrometer. Proteins present in lane 3 were subtracted from those identified in lane 1 to reduce the complexity of the mass spectrometry data set.</p

Immunostaining of muscle tissue with serum-derived IgG.

<p>Immunostaining of mouse muscle tissue demonstrates IBM-derived IgG positively label muscle tissue sections (A–I). The identity of the IgG specimens is shown in the top right corner of each panel. Staining is present within the endomysial space (green). Nuclei are stained in blue. In sections (A, C, D, F, G, H and I) IBM-derived IgG labeling can be seen in the endomysial space. In six of these samples binding was also observed in the perimysium (C, D and F–I). Tissue, from adjacent sections, stained with IgG derived from healthy donor (HD) serum (J–L) is free of any labeling.</p