Anu Sooda

Objective
Inclusion body myositis (IBM) is a progressive late-onset muscle disease characterised by preferential weakness of quadriceps femoris and finger flexors, with elusive causes involving immune, degenerative, genetic and age-related factors. Overlapping with normal muscle ageing makes diagnosis and prognosis problematic.

Methods
We characterised peripheral blood leucocytes in 81 IBM patients and 45 healthy controls using flow cytometry. Using a random forest classifier, we identified immune changes in IBM compared to HC. K-means clustering and the random forest one-versus-rest model classified patients into three immunophenotypic clusters. Functional outcome measures including mTUG, 2MWT, IBM-FRS, EAT-10, knee extension and grip strength were assessed across clusters.

Results
The random forest model achieved a 94% AUC ROC with 82.76% specificity and 100% sensitivity. Significant differences were found in IBM patients, including increased CD8+ T-bet+ cells, CD4+ T cells skewed towards a Th1 phenotype and altered γδ T cell repertoire with a reduced proportion of Vγ9+Vδ2+ cells. IBM patients formed three clusters: (i) activated and inflammatory CD8+ and CD4+ T-cell profile and the highest proportion of anti-cN1A-positive patients in cluster 1; (ii) limited inflammation in cluster 2; (iii) highly differentiated, pro-inflammatory T-cell profile in cluster 3. Additionally, no significant differences in patients' age and gender were detected between immunophenotype clusters; however, worsening trends were detected with several functional outcomes.

Conclusion
These findings unveil distinct immune profiles in IBM, shedding light on underlying pathological mechanisms for potential immunoregulatory therapeutic development.

OBJECTIVES: Inclusion body myositis (IBM) is a progressive inflammatory-degenerative muscle disease of older individuals, with some patients producing anti-cytosolic 5'-nucleotidase 1A (NT5C1A, aka cN1A) antibodies. Human Leukocyte Antigens (HLA) is the highest genetic risk factor for developing IBM. In this study, we aimed to further define the contribution of HLA alleles to IBM and the production of anti-cN1A antibodies.

METHODS: We HLA haplotyped a Western Australian cohort of 113 Caucasian IBM patients and 112 ethnically matched controls using Illumina next-generation sequencing. Allele frequency analysis and amino acid alignments were performed using the Genentech/MiDAS bioinformatics package. Allele frequencies were compared using Fisher's exact test. Age at onset analysis was performed using the ggstatsplot package. All analysis was carried out in RStudio version 1.4.1717.

RESULTS: Our findings validated the independent association of HLA-DRB1*03:01:01 with IBM and attributed the risk to an arginine residue in position 74 within the DRβ1 protein. Conversely, DRB4*01:01:01 and DQA1*01:02:01 were found to have protective effects; the carriers of DRB1*03:01:01 that did not possess these alleles had a fourteenfold increased risk of developing IBM over the general Caucasian population. Furthermore, patients with the abovementioned genotype developed symptoms on average five years earlier than patients without. We did not find any HLA associations with anti-cN1A antibody production.

CONCLUSIONS: High-resolution HLA sequencing more precisely characterised the alleles associated with IBM and defined a haplotype linked to earlier disease onset. Identification of the critical amino acid residue by advanced biostatistical analysis of immunogenetics data offers mechanistic insights and future directions into uncovering IBM aetiopathogenesis.

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.