Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity

IntroductionInclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). MethodsWe investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. ResultsBlood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. ConclusionAltogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden

Idiopathic inflammatory myopathies: A review

Idiopathic inflammatory myopathy (IIM) is the umbrella term including dermatomyositis (DM), polymyositis (PM), overlap myositis (OM), sporadic inclusion body myositis (IBM) and necrotising autoimmune myopathy (NAM), also known as immunemediated necrotising myopathy. There is some debate as to whether PM exists as a discrete entity, or perhaps is an overly generalising term encompassing connective tissue disease associated myositis, or OM, and the previously poorly recognised NAM. As such, PM will not be covered in detail in this review. DM, OM and NAM all present similarly, with proximal weakness and elevated creatine kinase (CK) level. By contrast, IBM preferentially involves the long finger flexors and quadriceps, and presents with a normal or only mildly elevated CK. Developments in serological testing and imaging are shifting the diagnostic paradigm away from a reliance on histopathology. The therapeutic armamentarium for IIM continues to evolve, with intravenous immunoglobulin and rituximab proving to be successful for refractory disease. This review will provide a diagnostic algorithm for the clinician to help distinguish between IIM subtypes – with emphasis on clinical assessment, serology and imaging, as well as discussion of therapeutic options and escalation of immunotherapy

Muscle B mode ultrasound and shear-wave elastography in idiopathic inflammatory myopathies (SWIM): Criterion validation against MRI and muscle biopsy findings in an incident patient cohort

Background: B mode ultrasound (US) and shear wave elastography (SWE) are easily accessible imaging tools for idiopathic inflammatory myopathies (IIM) but require further validation against standard diagnostic procedures such as MRI and muscle biopsy. Methods: In this prospective cross-sectional study we compared US findings to MRI and muscle biopsy findings in a group of 18 patients (11 F, 7 M) with active IIM (dermatomyositis 6, necrotising autoimmune myopathy 7, inclusion body myositis 4, overlap myositis 1) who had one or both procedures on the same muscle. US domains (echogenicity, fascial thickness, muscle bulk, shear wave speed and power doppler) in the deltoid and vastus lateralis were compared to MRI domains (muscle oedema, fatty infiltration/atrophy) and muscle biopsy findings (lymphocytic inflammation, myonecrosis, atrophy and fibro-fatty infiltration). A composite index score (1–4) was also used as an arbitrary indicator of overall muscle pathology in biopsies. Results: Increased echogenicity correlated with the presence of fatty infiltration/atrophy on MRI (p = 0.047) in the vastus lateralis, and showed a non-significant association with muscle inflammation, myonecrosis, fibrosis and fatty infiltration/atrophy (p \u3e 0.333). Severe echogenicity also had a non-significant association with higher composite biopsy index score in the vastus lateralis (p = 0.380). SWS and US measures of fascial thickness and muscle bulk showed poor discrimination in differentiating between pathologies on MRI or muscle biopsy. Power Doppler measures of vascularity correlated poorly with the presence of oedema on MRI, or with inflammation or fatty infiltration on biopsy. Overall, US was sensitive in detecting the presence of muscle pathology shown on MRI (67–100%) but showed poorer specificity (13–100%). Increased echogenicity showed good sensitivity when detecting muscle pathology (100%) but lacked specificity in differentiating muscle pathologies (0%). Most study participants rated US as the preferred imaging modality. Conclusions: Our findings show that US, in particular muscle echogenicity, has a high sensitivity, but low specificity, for detecting muscle pathology in IIM. Traditional visual grading scores are not IIM-specific and require further modification and validation. Future studies should continue to focus on developing a feasible scoring system, which is reliable and allows translation to clinical practice

Muscle shear wave elastography, conventional B mode and power doppler ultrasonography in healthy adults and patients with autoimmune inflammatory myopathies: A pilot cross-sectional study

Background: Before the role of shear wave elastography (SWE) and B mode ultrasound (US) in the diagnosis of different forms of idiopathic inflammatory myopathies (IIM) can be investigated, normative data is required. This study aimed to describe and then compare normative SWE and B mode ultrasound metrics of muscles in healthy controls and patients with IIM. Methods: Twenty nine healthy adult controls and 10 IIM patients (5 with inclusion body myositis and 5 with necrotising autoimmune myopathy) underwent a full clinical examination, laboratory investigations, SWE and US measurements of selected proximal and distal limb muscles. Shear wave speed (SWS) and multiple US domains [echogenicity, fascial thickness, muscle bulk and power Doppler (PD)] were measured in both groups. Results: In healthy controls (n = 29; mean age 46.60 ± 16.10; 44.8 % female), age was inversely correlated with SWS at the deltoid (stretch) (Rs. -0.40, p = 0.030) and PD score at the deltoid (rest) (Rs. -0.40, P = 0.032). Those ≥ 50 years old had a lower SWS at the deltoid (stretch) compared to the \u3c 50 year group (2.92 m/s vs. 2.40 m/s, P = 0.032). Age correlated with increased echogenicity in the flexor digitorum profundus (Rs. 0.38, P = 0.045). Females had a smaller muscle bulk in the deltoid (P = 0.022). Body mass index (BMI) was inversely associated with SWS in the deltoid (stretch) (Rs – 0.45, P = 0.026), and positively correlated with echogenicity in the deltoid (Rs. 0.69, P = 0.026). In patients ≥50 years of age, patients with IIM (mean age 61.00 ± 8.18; females 20.0 %) had a higher proportion of abnormal echogenicity scores at the flexor digitorum profundus (FDP) (40.00 % vs. 14.30 %, P = 0.022) and tibialis anterior (TA) (80.00 % vs. 28.60 %, P = 0.004). Fascial thickness was lower in the FDP (0.63mm vs. 0.50mm, p = 0.012) and TA (0.58mm vs. 0.45mm, P = 0.001). Conclusions: Our findings suggest there is scope for US techniques to be useful for diagnostic screening of affected muscles in patients with IIM, especially in those with suspected inclusion body myositis or necrotising autoimmune myopathy. We provide normative data for future studies into SWE and US techniques in skeletal muscle. The differences between IIM patients and controls warrant further study in a broader IIM patient cohort

Isolation of live leukocytes from human inflammatory muscles

In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further investigation. To gain access to live and isolated cells, many studies utilized blood samples; however, in the absence of biological tools to discriminate the leukocytes associated with the autoimmune process from those that emerged from responses against pathogens, the information observed on circulating immune cells often lacks in specificity, and thus result interpretation may prove difficult. In order to selectively retrieve self-reactive immune cells, we developed a protocol to isolate live leukocytes from human muscle biopsies, which allows for further analysis using a large range of methodologies. The protocol uses enzymatic digestion to release live leukocytes from freshly collected skeletal muscle samples, followed by filtration and separation of the leukocytes from the myocytes by density gradient centrifugation. The isolated cells can be submitted immediately to various analysis strategies to characterize ex vivo the specific cellular and molecular mechanisms responsible for self-directed immune muscle aggression or may be placed in culture for expansion

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity

Introduction: Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). Methods: We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Results: Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5\u27-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Conclusion: Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden