Jerome Coudert

Background. Inclusion body myositis (IBM) is the most common acquired skeletal muscle disease associated with ageing and involves both an autoimmune attack on muscle fibres as well as muscle degeneration with protein deposition. There have been multiple studies showing a relationship between exercise and improved outcomes in IBM, however only one prior trial showed a modest benefit of Oxandrolone in IBM. No studies have explored a combination of the two. We hypothesised that a combination of exercise training and testosterone treatment would improve muscle strength and physical activity in males affected by IBM, more than exercise alone.

Methods. This pilot study adopted a double-blinded, placebo-controlled, crossover design to assess whether testosterone combined with a prescribed exercise program would improve measures of muscle strength, physical activity and quality of life in men affected by IBM, over exercise alone. Treatment (exercise and placebo) and placebo (exercise only) arms were 12 weeks in duration, with a two-week wash-out period between arms. Primary outcome was quadriceps strength, measured by isokinetic dynamometer (Humac Norm). Secondary outcomes included lean body mass, functional tests and Patient Reported Outcomes (PROs). Outcome measures were collected at baseline, Week 12 and Week 26. In response to participant feedback, a 12-month Open Label Extension (OLE) was offered, with outcome measures collected at new baseline, 6-months and 12-months.

Results. The primary outcome, quadriceps extension strength the placebo and treatment arms, was non-significant. The DEXA assessment of lean body mass and other functional tests were also non-significant between the placebo and treatment arms. There was a significant reduction of peak torque extension at 60 degrees during the placebo period (p=0.04). Within the SF-36 questionnaire, the domain of ‘role limitations due to emotional problems’ demonstrated higher scores during the testosterone arm when compared to the placebo arm (p=0.044). All other domains, as well as the IBM-FRS, were non-significant. Within the OLE, the 2- minute walk test regular and fast were significant at 6 months (p=0.03 and p=0.032 respectively) but not at 12 months. The most common adverse event reported was falls which were considered a complication of IBM rather than the study. Within the OLE, 3 of the 12 participants withdrew from the study due to adverse events related to rising prostate specific antigen and haematocrit.

Conclusions. In this study comparing exercise alone to exercise in combination of testosterone supplementation, we were unable to detect a significant effect of the treatment on muscle strength, function or quality of life. However, peak torque extension at 60 degrees worsened significantly during the exercise-only arm, which could indicate that testosterone conferred some stabilisation effect during the treatment period. Individuals on testosterone also felt that their daily activities were not as limited due to the emotional problems compared with placebo, perhaps indicating that testosterone contributes to improved mood and mental health. Individuals who participated in the OLE had improved walk tests at 6 months which then declined at 12 months. This warrants further investigation in future studies, where the benefit of testosterone on walking strength may decline after a prolonged period. Finally, we note that 12 men requested the OLE due to perceived positive effects and lack of adverse effects, so potentially the benefits of testosterone require a longer period of intervention to manifest than that applied during the crossover phase.

Background. Inclusions of Sequestosome1 (SQSTM1/p62) within muscle fibers are a pathological hallmark of sporadic inclusion body myositis (sIBM) with p62 overexpression reported in patients. Mounting evidence suggests a role for p62 expression and/or variation in sIBM pathology, due to the presence of rare and potentially pathogenic missense variants (A117V, G194R, K238E, P392L). Consequently, we hypothesized that genetic modifiers of SQSTM1 may present a critical missing link for sIBM pathology and contribute to disease expres¬sion. Short structural variants (SSVs) are a class of genetic variation that can be difficult to characterized due to their highly repetitive and complex nature. Evidence that SSVs play an important role in complex diseases such as Alzhei¬mer’s Disease, Amyotrophic lateral sclerosis, Spinocerebellar Ataxia type 2, and Huntington’s disease is now confirmed and further investigations of this type of genetic variation is necessary to uncover missing heritability in complex dis¬eases. We and others have previously reported an SSV within SQSTM1 that is associated with altered expression of p62. The SSV rs60327661 is a CAAA in¬sertion/deletion within intron 5 of SQSTM1, which also confers risk for familial Amyotrophic lateral sclerosis. Due to the role of the SSV in Amyotrophic lateral sclerosis and altered p62 expression, we hypothesized the SSV rs60327661 may have disease-modifying effects in a longitudinal cohort of sIBM patients.

Methods. DNA samples from 218 sIBM patients and 242 healthy controls were received from The Institute for Immunology and Infectious Diseases, Murdoch, Western Australia, and the NINDS Repository, Coriell Institute for Medical Research, New Jersey. Genomic DNA samples were systematically assessed through polymerase chain reaction, capillary separation, and Sanger sequencing for rs60327661 allele genotyping.

Results: In the present study, when controlling for self-declared ancestry, car¬riage of the D/D genotype is associated with sIBM disease expression (p<0.05). Both the case and control groups did not violate Hardy-Weinberg equilibrium (p=0.99, p=0.98; respectively. Intriguingly, patients who were CN1A seroposi¬tive were more likely carry the D allele (n=18) when compared to patients with-out a D allele (n=3; p<0.047). Patients classified as fast progressors (n=2) carried only the D/D genotype.

Conclusion. In this study, we present the first report of an association between the SQSTM1 insertion/deletion and sIBM disease expression. We provide evi¬dence that the investigation of genetic variants outside of the HLA region is war¬ranted, and that such investigations are likely to uncover critical information for sIBM. We present the SSV rs60327661 as a novel disease modifying variant for sIBM which is functionally linked to p62 by altering protein expression. Our data adds to growing evidence that examination of SSVs may uncover novel genetic risk markers, and consequently further understanding of the pathogenic mechanisms at play.

Background. Idiopathic Inflammatory Myopathies (myositis) are a group of rare neuromuscular diseases. Conditions within this group include Dermatomyositis (DM), Necrotising Myositis (NAM), Polymyositis (PM), Overlap Myositis and the currently untreatable Inclusion Body Myositis (IBM). It is well established that patients within research-active clinical environments have better outcomes and development of effective disease-modifying treatments is dependent on translational, patient-centered research.

Aim: Establishment of a translational research programme within specialist myositis outpatient clinics at Perron Institute for Neurological and Translational Science and Murdoch University (Perth, Australia).

Methods. Facilitated through a significant patient bequest towards myositis re¬search, a laboratory and clinical research programme was founded in 2017 by Professor Merrilee Needham. Appointment of laboratory and clinical research leads supported new workflows where during clinic visits, patients meet with research nurses/coordinators and are provided the opportunity to take part in myositis research, including donating blood and urine samples that are taken directly to the on-site laboratories for analysis. Consented patients are also enrolled in the team’s emerging observational Myositis Registry, and natural history data is collected via questionnaires and outcome measures through our team physiotherapist. At their clinic visit, patients are kept up to date with clinical trial opportunities and research news. For those actively taking part in clinical trials, re¬search visits are often aligned with clinic appointments, increasing convenience and ease of participation. Within our laboratory programme, Lead Scientist Dr Jerome Coudert and his team are characterising the immune profiles, underlying pathways and mechanisms, genetic and metabolic changes of patients over time, as well as performing detailed analyses on blood, urine and muscle samples. A critical element of the research clinic model is keeping patients actively engaged with the research programme. Our patients are kept up to date with current and upcoming projects, advise us of what is important to study next, and are actively participating in project design and review. In recent years, our group has been complimented by an increasing number of higher degree students as well as medical students, across both the clinical and laboratory arms of the programme.

Results. At time of submission, we have over 400 active patients and disease-control participants, with over 2,000 samples collected and biobanked. Our ethical approvals support invitation of participants to take part in future studies based on genotype or phenotype of interest. Our programme has supported five Investigator-Initiated clinical trials over the last 3 years and four commercial clinical trials in myositis. Our highly collaborative approach sees growing national and international collaborative projects and grant applications. In 2020 we received a $1.8M grant from the Australian Government to lead an international, multi-site, Phase 3 trial of Sirolimus in IBM.

Conclusions. Research allows us to offer our patients hope and to partner with them on the journey to finding new treatments and improving quality of life. Our translational programme features a direct and bi-directional pipeline between laboratory and clinical research, built on a solid foundation of observational research projects designed to facilitate future research and treatment trials in myositis.

Objectives. Inclusion body myositis (IBM) is a progressive inflammatory and degenerative disease of the skeletal muscles that affects individuals over the age of 45 and leads to a gradual loss of mobility. It has been widely reported that a subgroup of 33 to 72% of IBM patients produce self-reactive antibodies that bind cytosolic 5’-nucleotidase 1A (NT5C1A) within the muscles and possibly exacerbate the disease severity. A genetic association between immune-related genes with IBM has been described. This study aimed to deepen our knowledge about the human leukocyte antigen (HLA) genes that alter the risk to develop IBM and to investigate whether specific HLA alleles may contribute to the occurrence of NT5C1A-directed antibodies.

Methods. In this study, we used Illumina next-generation sequencing to resolve the high resolution HLA haplotype of 102 Caucasian IBM patients from the Western Australian cohort. We then compared the frequency and carriage of the identified alleles within the IBM cohort to reference databases of Caucasian cohorts. We additionally compared the HLA allele carriage within the genotypes of anti-NT5C1A-positive and-negative patients within our IBM cohort.

Results. Our results confirmed the previously identified association risk of the 8.1 MHC ancestral haplotype with IBM. We also lifted ambiguities and clarified the identity of the risk-associated alleles that have been previously reported at a lower level of resolution. Additionally, we identified previously unreported risk allele associations with IBM. Furthermore, our analysis validated previously reported protective HLA alleles, and also revealed reduced carriage frequency of additional alleles, suggesting their protective role in the disease. Lastly, our study revealed two alleles, which carriage are associated with anti-NT5C1A antibody production.

Conclusions. Our findings refine and expand on the knowledge of the HLA genetic background of IBM. Stratifying patients based on their HLA genotype provides a genetic basis for new therapeutic intervention strategies early in the disease process to slow down symptom development.

Background. Inclusion body myositis (IBM) is the most common inflammatory muscle diseases that primarily affects the elderly. It is characterised by autoim¬mune aggression and degeneration of skeletal muscles, which leads to severe disability over time. Although the aetiology of IBM is uncertain, numerous lines of evidence point to T cells playing a pathogenic key role. One of the major ques¬tions that remains unsolved regarding IBM muscle-invading T cells is the nature of the antigen that drives their autoreactivity. We hypothesised that the analysis of T cells’ T cell receptor (TCR) repertoire will give insights into their specific¬ity. We used high-throughput sequencing of TCRβ chains in T cells isolated from muscle biopsies and matched blood samples to compare T cell clonal profiles within the inflammation-affected muscle tissues and systemically.

Methods. Blood and muscle samples were collected on the same days for each of the four donors. Muscle-invading T cells were isolated as bulk while peripheral blood mononuclear cells were subjected to CD4+ and CD8+ T cell separation. The TCRβ sequencing was performed using the high-throughput Illumina Miseq platform. Downstream data analysis of TCRβ repertoires was performed using in-house VGAS tool, Immunarch R package and VDJtools. HLA haplotype of each donor was determined by high resolution typing of HLA class I and II al¬leles using Illumina Miseq platform in an American Society for Histocompat¬ibility and Immunogenetics (ASHI)-accredited laboratory.

Results. Analysis of muscle-T cells for TCR repertoire overlap revealed shared clonotypes between patients. A unique TCRβ sequence was shared between pa¬tients 1, 3, and 4, while in patient 2, although this sequence was not found in the muscle, it ranked as a predominant clone in the blood. The patients 1 and 3 displayed nine sequences in common, as well as their top three TCRβ sequences. Furthermore, analysis of TCR repertoire usage in the corresponding blood sam¬ples showed common clonotypes between muscle and blood, but at higher fre¬quency in muscles indicating that a preferential expansion occurred in this tissue. Moreover, querying the top five dominant TCRβ CDR3 sequences of muscle from each patient in a curated database of TCR identified multiple highly similar sequences with known specificity for antigens derived from virus and muscle proteins, especially in the CD8+ T cell subset. In patient 1 and 3, the topmost clone showed a high level of similarity with a CDR3 specific for CMV-derived antigen as well as self-antigens derived from protein phosphatase 1F (PPM1F) and A-kinase anchor protein 9 (AKAP9). The HLA alleles reported to present these autoantigens matched with the HLA haplotype of patient 3. On the other hand, for patient 2, we identified a clone bearing high CDR3 similarity with known sequences documented to be associated with HIV-1 and EBV-derived an¬tigens, and another CDR3 that exactly matched a sequence specific for a gluten-derived peptide. Interestingly, this patient carries HLA-DQ9 which is a reported risk factor for celiac disease.

Conclusions. Our findings identified public TCRs in IBM muscle, and the pres¬ence of expanded T cell clones harbouring TCR sequences with striking similari¬ties between virus and muscle-derived antigens, suggesting that an underlying molecular mimicry mechanism may generate autoreactive T cells.

Background. Inclusion body myositis (IBM) is an autoimmune disease characterised by intense muscle infiltration by CD8+ T lymphocytes with a predominant terminally differentiated effector (TEMRA) phenotype. Recently, IBM was found to be often associated with a lymphoproliferative disorder of CD8+ T cells known as T cell Large Granular Lymphocytes (T-LGL). T-LGLs represent a spectrum of conditions that range from a lymphoproliferation in response to antigenic stimulation “reactive” to an aggressive leukemia. The incidence of expanded T-LGLs among IBM patients (between 40-60%) suggests that these cells play a distinct, yet unknown role in the pathology of this disease. This study aims to determine the T-LGL phenotypical characteristics and TCR clonal diversity and investigates the clinical implications of T-LGLs in IBM.

Methods. Blood samples collected from 85 IBM patients and an aged-matched group of 56 healthy donors were analysed using flow cytometry for phenotype characterisation. CD8+ T cells isolated from PBMCs collected in each donor group were separated into CD57+ (T-LGL) and CD57- (internal control) and sequenced to determine TCR repertoire diversity in both cell type. Multi-variate analysis of biological and clinical data at the time of sample collection, such as aid-assisted mobility, was used to investigate the impact of T-LGL in disease severity.

Results. 33/85 (39%) of IBM patients and 6/56 (10.7%) of healthy controls (HC) exhibited an elevated number of T-LGLs in blood. Immunophenotyping of the TCRαβCD8+ T-LGL population revealed aberrant expression of surface molecules such as decreased CD5 and increased CD57+, CD56+, and KLRG1+ that are commonly displayed by the natural killer cell lineage. Longitudinal analysis of thirteen T-LGL+ patients showed that this expanded cell population persists over time (average time elapsed between samples=1.5 years). Comparison of the proportion of T-LGL within the CD8 T cell population between muscle and blood samples showed that they preferentially accumulate within the muscle (blood: mean= 41.1%±11.7% vs Muscle mean= 70.1%±12.8%). Blood T-LGLs isolated from 6 IBM and 2 HC donors and submitted to bulk TCR-analysis consistently exhibited polyclonal characteristics, with diverse TCRβ VDJ segment-usage and CDR3 repertoire. Clinically, an increased proportion of IBM T-LGL positive patients required more-assisted mobility aids compared to T-LGL negative patients, even though both groups had experienced IBM symptoms for similar durations (T-LGL+ median=12 years vs T-LGL- median=11 years, p-value=0.99)

Conclusions. Our results demonstrate that a CD8+ T-LGLs lymphoproliferative disorder is more prevalent in IBM patients than in healthy individuals. These cells display a late-differentiated phenotype, characterised by reduced TCR-associated co-receptors and high expression of innate lymphocyte-related surface co-receptors and adhesion molecules, suggesting that these cells may be submitted to TCR-independent regulation. In contrast with previous findings, we found that the peripheral CD8+ T-LGL expansion is polyclonal suggesting their expansion is reactive in nature, yet capable of persisting for years. Nonetheless, the presence of expanded CD8+ T-LGL is associated with increased disease severity in IBM. Further investigations into the clonal diversity of muscle-infiltrating T-LGLs should be evaluated to determine if their expansion is antigen-driven or due to bystander activation.

Background. Sporadic Inclusion body myositis (IBM) is an inflammatory disease that affects skeletal muscles in individuals over the age of 45. It leads to progressive muscle wasting and progressive disability and loss of independence for patients. Histologically, inflammation is characterised by myofibers displaying upregulated expression of major histocompatibility complex (MHC) class I molecules and invasion by immune cells comprising abundant CD8+ T cells. There is currently no cure, and regular exercise is the only recommended treatment recognised to be effective at limiting muscle inflammation and hindering the dis¬ease progression. In healthy men, physical exercise along with administration of testosterone was shown to increase skeletal muscle mass and performance in an additive fashion. Previous studies report that androgens are anti-inflammatory, inhibiting CD4+ T cell differentiation into inflammatory Th1 cells and promoting regulatory T cells. We hypothesised that combining testosterone supplementation with exercise training will reduce the level of autoimmune inflammation better than exercise alone, in men affected by IBM.

Methodology. We conducted a double-blind, placebo-controlled, cross-over randomised controlled trial (RCT) in fourteen men with IBM, to assess whether a period of exercise training using a personalised regime combined with topical application of testosterone, reduced the inflammatory immune response associated with this disease over and above just exercise alone. To assess the intervention efficacy, we analysed the phenotype of immune cells in the blood by flow cytometry, and measured the serum cytokine and chemokine content by Luminex immunoassay.

Results. The testosterone supplementation resulted only in a significant reduction of eosinophil numbers. However, we found additional immunoregulatory effects of the exercise training program over study that were testosterone-independent, including altered proportions of subsets within monocytes, T and B cells, and reduced circulating concentration of several pro-inflammatory cytokines such as IL-12, IL-17, TNF-alpha, MIP-1beta and sICAM-1.

Conclusions. Overall, our findings indicate that regular exercise training impacts the immune response in IBM and provides anti-inflammatory benefits to IBM patients; concomitant testosterone supplementation over a 12-week period provided essentially no anti-inflammatory effect over exercise alone. This work further emphasizes that maintaining a regular level of exercise helps in controlling inflammation in IBM, and also possibly, the pace of progression of the disease toward severe stages. We cannot exclude that incorporating testosterone supple-mentation to exercise training may provide a synergistic anti-inflammatory in IBM, but the optimal duration of intervention and the stage of disease progression when the intervention provides most benefits will need to be further characterised and refined to achieve optimal outcomes.