Olivier Benveniste

Efficacy of Rituximab in Refractory Inflammatory Myopathies Associated with Anti- Synthetase Auto-Antibodies: An Open-Label, Phase II Trial.

Objective Anti-synthetase syndrome (anti-SS) is frequently associated with myositis and interstitial lung disease (ILD). We evaluated prospectively, in a multicenter, open-label, phase II study, the efficacy of rituximab on muscle and lung outcomes. Methods Patients were enrolled if they were refractory to conventional treatments (prednisone and at least 2 immunosuppressants). They received 1 g of rituximab at D0, D15, and M6. The primary endpoint was muscular improvement based on manual muscular testing (MMT10, Kendall score in 10 muscles) at M12. Secondary endpoints were normalization of creatine kinase (CK) level, ILD improvement based on forced vital capacity and/or diffuse capacity for carbon monoxide, and number and/or doses of associated immunosuppressants. Results Twelve patients were enrolled, and 10 completed the study. Only 2 patients presented an improvement of at least 4 points on at least two muscle groups (primary end-point). Overall, seven patients had an increase of at least 4 points on MMT10. CK level decreased from 399 IU/L (range, 48–11,718) to 74.5 IU/L (range, 40–47,857). Corticosteroid doses decreased from 52.5 mg/d (range, 10–70) to 9 mg/d (range, 7–65) and six patients had a decrease in the burden of their associated immunosuppressants. At baseline, all 10 patients presented with ILD. At M12, improvement of ILD was observed in 5 out of the 10 patients, stabilization in 4, and worsening in 1. Conclusions This pilot study of rituximab treatment in patients with refractory anti-SS provided data on evolution of muscular and pulmonary parameters. Rituximab should now be evaluated in a larger, controlled study for this homogenous group of patients. Trial Registration Clinicaltrials.gov NCT00774462.

Innate and Adaptive Immune Response in Fabry Disease

Fabry disease is an X-linked lysosomal storage disease in which mutations of the gene (GLA) cause a deficiency of the lysosomal hydrolase α-galactosidase A (α-Gal). This defect results in an accumulation of glycosphingolipids, primarily globotriaosylceramide (Gb3) which causes a multisystemic vasculopathy. Available since 2001 in Europe, enzyme replacement therapy consists in the administration of agalsidase, a recombinant form of α-galactosidase A. Enzyme replacement therapy was shown to improve the global prognosis but allowed partial success in preventing critical events such as strokes and cardiac arrests. As in most lysosomal storage diseases, frequent immune reactions have been described in naive Fabry disease patients. Humoral immune responses following enzyme replacement therapy have also been described, with unclear consequences on the progression of the disease. While cost-effectiveness of enzyme replacement therapy in Fabry disease begins to be questioned and new therapeutic strategies arise such as chaperone or gene therapy, it appears necessary to better understand the immune responses observed in the treatment of naive patients and during enzyme replacement therapy with agalsidase. We propose a comprehensive review of the available literature concerning both innate and adaptive responses observed in Fabry disease. We particularly highlight the probable role of the toll-like receptor 4 (TLR4) and CD1d pathways triggered by Gb3 accumulation in the development of local and systemic inflammation that could lead to irreversible organ damages. We propose an immunological point of view of Fabry disease pathogenesis involving immune cells notably the invariant natural killer T cells. We finally review anti-agalsidase antibodies, their development and impact on outcomes.

The immunoproteasomes are key to regulate myokines and MHC class I expression in idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are diseases with muscle weakness, morphologically characterized by inflammatory infiltration and increased expression of MHC class I molecule on myofibers. Immunoproteasome, as a proteolytic complex that shapes the repertoire of antigenic peptides, has been previously demonstrated to be over-expressed in IIMs at mRNA level. In this study, we investigated the expression and the function of the immunoproteasome in IIMs in more detail. As shown by immunofluorescence staining, expression of relevant players of the immunoproteasome was detectable in the inflamed skeletal muscle tissue from IIM patients. In fact, two subunits of the immunoproteasome, β1i or β5i were upregulated in sporadic inclusion body myositis, immune-mediated necrotizing myopathies and dermatomyositis muscle biopsies and co-localized with the MHC class I expressing myofibers. Double immunofluorescence revealed that both myofibers and muscle infiltrating cells, including CD8+ T-cells and CD68xa0+xa0macrophages in IIMs expressed β1i or β5i. In addition, we have also investigated the role of the immunoproteasome in myoblasts during inxa0vitro inflammatory conditions. Using human primary myoblasts cultures we found that pro-inflammatory cytokines, TNF-α or IFN-γ upregulate β1i or β5i. Selective inhibition or depletion of β5i amplified the TNF-α or IFN-γ mediated expression of cytokines/chemokines (myokines) in myoblasts. Furthermore, we demonstrated that specific inhibitors of β1i or β5i reduced the cell surface expression of MHC class I in myoblasts induced by IFN-γ. Taken together, our data suggest that the immunoproteasome is involved in pathologic MHC class I expression and maintenance of myokine production in IIMs. Thus, induction of the immunoproteasome was identified as a pathomechanism underlying inflammation in IIMs.

Development of a New Classification System for Idiopathic Inflammatory Myopathies Based on Clinical Manifestations and Myositis-Specific Autoantibodies

Importance Idiopathic inflammatory myopathies are heterogeneous in their pathophysiologic features and prognosis. The emergence of myositis-specific autoantibodies suggests that subgroups of patients exist. Objective To develop a new classification scheme for idiopathic inflammatory myopathies based on phenotypic, biological, and immunologic criteria. Design, Setting, and Participants An observational, retrospective cohort study was performed using a database of the French myositis network. Patients identified from referral centers for neuromuscular diseases were included from January 1, 2003, to February 1, 2016. Of 445 initial patients, 185 patients were excluded and 260 adult patients with myositis who had complete data and defined historical classifications for polymyositis, dermatomyositis, and inclusion body myositis were enrolled. All patients were tested for anti–histidyl-ARN-t- synthetase (Jo1), anti–threonine-ARN-t-synthetase (PL7), anti–alanine-ARN-t-synthetase (PL12), anti–complex nucleosome remodeling histone deacetylase (Mi2), anti-Ku, anti–polymyositis/systemic scleroderma (PMScl), anti–topoisomerase 1 (Scl70), and anti–signal recognition particle (SRP) antibodies. A total of 708 variables were collected per patient (eg, cancer, lung involvement, and myositis-specific antibodies). Main Outcomes and Measures Unsupervised multiple correspondence analysis and hierarchical clustering analysis to aggregate patients in subgroups. Results Among 260 participants (163 [62.7%] women; mean age, 59.7 years; median age [range], 61.5 years [48-71 years]), 4 clusters of patients emerged. Cluster 1 (nu2009=u200977) included patients who were male, white, and older than 60 years and had finger flexor and quadriceps weakness and findings of vacuolated fibers and mitochondrial abnormalities. Cluster 1 regrouped patients who had inclusion body myositis (72 of 77 patients [93.5%]; 95% CI, 85.5%-97.8%; Pu2009<u2009.001). Cluster 2 (nu2009=u200991) regrouped patients who were women and had high creatine phosphokinase levels, necrosis without inflammation, and anti–SRP or anti–3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies corresponding to immune-mediated necrotizing myopathy (53 of 91 [58.2%]; 95% CI, 47.4%-68.5%; Pu2009<u2009.001). Cluster 3 (nu2009=u200952) regrouped patients who had dermatomyositis rash and anti-Mi2, anti–melanoma differentiation-associated protein 5 (MDA5), or anti–transcription intermediary factor-1&ggr; (TIF1&ggr;) antibodies, mainly corresponding with patients who had dermatomyositis (43 of 52 [82.7%]; 95% CI, 69.7%-91.8%; Pu2009<u2009.001). Cluster 4 (nu2009=u200940) was defined by the presence of anti-Jo1 or anti-PL7 antibodies corresponding to antisynthetase syndrome (36 of 40 [90.0%]; 95% CI, 76.3%-97.2%; Pu2009<u2009.001). The classification of an independent cohort (nu2009=u200950) confirmed the 4 clusters (Cohen &kgr; light, 0.8; 95% CI, 0.6-0.9). Conclusions and Relevance These findings suggest a classification of idiopathic inflammatory myopathies with 4 subgroups: dermatomyositis, inclusion body myositis, immune-mediated necrotizing myopathy, and antisynthetase syndrome. This classification system suggests that a targeted clinical-serologic approach for identifying idiopathic inflammatory myopathies may be warranted.

523. Deep Sequencing of T Cell Receptor in Peripheral Blood and Muscle from Adeno-Associated Virus Vector-Injected Subjects Reveals Differences in T Cell Clonality Between the Two Compartments

Long-term correction of the disease phenotype following gene therapy in vivo with adeno-associated virus (AAV) vectors has been demonstrated in several preclinical and clinical studies. With clinical development, however, it has become obvious that the host immune system represents an important obstacle to safe and effective gene transfer with AAV vectors. Indeed T cell responses directed against the viral capsid can have a detrimental effect on the duration of transgene expression and can result in tissue-specific toxicity due to the clearance of vector infected cells. ELISpot and flow cytometry analysis of immune responses to the vector capsid in peripheral blood mononuclear cells (PBMCs) show that both CD4+ and CD8+ T cells reactive to the AAV capsid are detectable in peripheral blood following gene transfer in humans, however little is known about T cell clonality following gene transfer in PBMC vs. locally in the tissue in which the vector is delivered.Here we used TCRbeta deep sequencing of T cells infiltrating muscle injected with AAV vectors and correlated results with a time course of sequencing of TCRbeta performed in fractionated CD8+ and CD8- PBMC from subjects injected with an AAV1 vector expressing γ-sarcoglycan (AAV1-γ-sarco). PBMC and muscle samples from three subjects who received an intramuscular injection of 4.5×1010 vector genomes of AAV1-γ-sarco (Brain 2012; 135:483) were analyzed. In one of these subjects, AAV capsid IFN-γ ELISpot on PBMCs showed activation of T cells responses peaking at day 30 post gene delivery. Consistent with this finding, TCRbeta sequencing of sorted CD8+ T cells showed the de novo expansion of T cell clones in PBMC after gene transfer, which were detectable at day 30 and 180 but not at baseline before vector administration. Additional CD8+ T cell clones present at baselines also expanded between day 0 and day 180. Interestingly, CD8+ T cell clones that expanded in PBMC following gene transfer were not found in biopsies of injected muscle collected at day 30. Conversely, several T cell clones shared between muscle and more in PBMC CD8+ than CD8- population did not expand upon AAV vector injection, possibly representing the underlying inflammation of muscle characteristic of the disease. Finally, we found T cell clones over-represented in muscle following AAV injection compared to PBMC, suggesting specific differences in homing of T cells in different body tissues. For example in one subject, we observed a clear expansion of T cell clones in muscle at day 30 post vector administration compared to baseline; these clones were not detected in the CD8+ T cell compartment in peripheral blood.In conclusion, this work addresses the crucial issue of correlation of T cell responses to AAV in peripheral blood vs. the transduced tissue. Our results suggest that T cell reactivity in peripheral blood does not necessarily represent the state of inflammation in the injected tissue, at the site of antigen presentation.