Background: Systemic Sclerosis (dcSSc) is an autoimmune disorder that affects the connective tissue. It is characterized by the thickening of the skin, which can lead to the fibrosis of internal organs. Although the overactivation of the immune system and increased proinflammatory cytokine production can explain the pathophysiology of this disease to a certain extent, the mechanisms leading to this are not well understood. Checkpoint inhibitory molecules function as physiological brakes in the immune system and help to maintain homeostasis. Aberrant checkpoint inhibitory pathways are often observed in patients with dcSSc. Lymphocyte activation gene-3 (LAG-3) is a late checkpoint inhibitory receptor that is mainly expressed by T cells and is involved in T cell inhibition. In this study, we focused on the role of LAG-3 in dcSSc and the effect of a LAG-3 agonist on immune cells from these patients. Objectives: This study aims to analyse the role of LAG-3 dcSSc and the influence of a LAG-3 Agonistic antibody in influencing inflammation and fibrosis. Methods: Plasma LAG-3 levels of 36 patients with dcSSc were compared to 20 healthy controls. PBMC’s from eight dcSSc patients and four Healthy Controls were analysed by flow cytometry for the surface expression of LAG-3 on CD4+ and CD8+T cells after stimulation with CD3/CD28 for 24 hours. Eight dcSSc PBMC’s were stimulated for 48 h with a LAG-3 Agonistic antibody and an isotype control. The harvested supernatants were analysed using a multiplex proinflammatory cytokine ELISA. Results: Patients with dcSSc had decreased soluble levels of LAG-3 compared with healthy controls (p <0.0001). Increased surface expression of LAG-3 was observed in CD4+ T cells(p=<0.05) and CD8+ T cells(p=<0.005) in patients with dcSSc compared to healthy control PBMCs. When treated with a LAG-3 agonistic antibody (LAG-3 Ag), PBMC monocultures from dcSSc patients significantly downregulated the production of IFN gamma, IL-2, IL-4 and TNF alpha and upregulated the production of IL-10 as compared to non-treated controls(NT) (*=p<0.05,**=p<0.005). The Isotype control did not show a similar effect. Conclusion: LAG-3 is immune-protective in Systemic Sclerosis and its agonistic antibody could be used as a potential treatment modality. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Figure 1

Background Pulmonary fibrosis is one of the major manifestations in Systemic Sclerosis (SSc) associated with high mortality. Mesenchymal transformation of the airway epithelial cells has been implicated as one of the causes for developing pulmonary fibrosis. Though several animal models shed light towards some of these aspects, an in vitro airway epithelial model would provide a novel experimental platform for the understanding and molecular and genetic changes that occur in SSc associated pulmonary fibrosis. Objectives To establish a functional model for airway epithelium from patient with diffuse cutaneous SSc (dSSc)and healthy volunteers derived nasal stem cells. Subsequently to induce Epithelial Mesenchymal transformation (EMT). Methods Nasal stem cells harvested from healthy volunteers(HV) and dSSc patients were differentiated into ciliated airway epithelium in an Air -Liquid Interface (ALI) using a transwell system. 4 HV cultures were then stimulated with TGF beta (5ug/ml) for 10 days at a basal stage and when differentiated. Markers of mesenchymal transformation including loss of E cadherin, and gain of N cadherin, fibronectin and vimentin were analysed by flow cytometry and image stream, and mean expression intensities given as (MFI). Secreted Type 1 collagen and fibronectin were measured by ELISA. Results Ciliated epithelial cultures could successfully be established from nasal stem cells (Figure 1). TGF beta induced a phenotypic change in the epithelial cells towards a mesenchymal one in HV cultures. This was observed by significantly increased expression of fibronectin and vimentin and loss of expression of E cadherin on the ciliated cells with 7 days of stimulation with TGF beta at a basal stage (Figure 1b). When cells, stimulated with TGF beta for 7 days, were analysed at Day 35 a similar trend was seen in their Delta MFI (Figure 1c). Stimulating the ALI cultures with TGF beta for 20 days completely repressed epithelial cell growth and disrupted their microstructure. Figure 1. Conclusion This novel ALI differentiated Airway epithelial model serves as a functional organoid to test various pulmonary manifestations of Systemic Sclerosis. The ability to induce Epithelial Mesenchymal Transformation of these cultures provides a proof of concept for TGF beta mediated fibrosis in dSSc. Moreover, this model can be utilized to explore, at the cell and molecular level, the impact of various autoantibodies and therapeutics on epithelial cells. References [1]Mehmet Kesimer,1 Sara Kirkham,2 Raymond J. Pickles,3 Ashley G. Henderson,4 Neil E. Alexis,5 Genevieve DeMaria,1 David Knight,2 David J. Thornton,2 and John K. Sheehan1 Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?; Am J Physiol Lung Cell Mol Physiol 296: L92–L100, 2009 Disclosure of Interests None declared.

Background Systemic Sclerosis (SSc) is a rare disorder associated with hand radiological damage, typically acro-osteolysis (AO) and calcinosis, and increased risk of osteoporosis [1, 2]. Recently, high resolution peripheral quantitative computed tomography (HR-pQCT) has contributed to advances in noninvasive imaging in patients with osteoporosis, inflammatory arthritis, and osteoarthritis. Due to its three-dimensional, high resolution (82 μm 3 ) imaging of calcified structures, HR-pQCT might be beneficial in detecting and describing early AO changes, calcifications or bone mineral density (BMD) loss and in studying potential treatment effects on progression/resolution of pathological manifestations. Objectives To study the association between different types of hand radiological damage assessed by HR-pQCT, and to evaluate volumetric BMD and microarchitecture at distal radius among SSc patients with and without AO. Methods This single-centre, cross-sectional study included patients with SSc according to ACR/EULAR 2013 recruited from the outpatient clinic at the Department of Rheumatology, Aarhus University Hospital, between 1st of October and 21st of December 2021. All patients underwent HR-pQCT (Xtreme CT, Scanco Medical AG) scan of the dominant hand’s distal radius and both hand’s distal phalanges (DP) on the 2 nd to 4 th finger. A semiquantitative scale of 0-3 was composed to quantify the degree of soft tissue calcifications identified in the 2 nd to 4 th finger (Figure 1). Lengths of the DPs and soft tissue extending distally from the most distal part of DPs (ST) were measured in a three-dimensional multiplanar reconstruction. Volumetric BMD and bone microarchitecture were analyzed using HR-pQCT scanner specific software at the distal radius. Figure 1. Results We included 40 patients with SSc of whom 13 had AO according to hand radiographs. The two groups had a similar distribution regarding age, sex, and BMI. Patients with AO had longer disease duration compared to patients without AO (med. 17 yrs. (IQR 7-20 yrs.) vs 3 yrs. (IQR 2-7 yrs.) ( P <0.001)). One patient was excluded from the BMD analysis, and 21 (9%) DP and 20 (8%) soft tissue measurements were excluded due to motion-induced image degradation. A higher prevalence of calcinosis was observed in patients with AO ( n =11/13) compared to patients without AO ( n =11/27) ( P <0.05). Patients with AO had more or larger calcifications according to our grading system (med. 2 (IQR 1-3) vs 0 (IQR 0-1)) ( P <0.001). Grade 3 changes were observed in patients with AO ( n =6/13, 46%) but not in patients without AO. Furthermore, the patients with AO had shorter DPs on the 2nd finger on both hands (right P<0.001; left P<0.01) and less ST on the right 2nd, 3rd, and 4th (P<0.01; P<0.001; P<0.01) as well as the left 3rd (P<0.05). We observed no differences between bone volume ratio (med. 0.101 (IQR 0.068-0.143) vs 0.126 (IQR 0.104-0.141) ( P =0.38)), cortical BMD (med. 828 (IQR 774-853) vs 842 (IQR 762-882) ( P =0.60)), or trabecular BMD (med. 121 (IQR 82-172) vs 151.6 (IQR 125-170) ( P =0.36)). Yet, the patients with AO had a lower number of trabeculae ( P <0.05) and a lower homogeneity of spacing between trabeculae ( P <0.05) at distal radius. Conclusion Patients with AO had larger and a higher number of calcifications and had significantly shortened DPs and ST on several fingers assessed by HR-pQCT. A potential superiority compared with hand radiographs is yet to be established. Still, the modality could eventually prove useful in detecting and monitoring small hand lesions and facilitate earlier diagnosis and proper treatment among patients with SSc. References [1]Koutaissoff, S., et al., Hand radiological damage in systemic sclerosis: comparison with a control group and clinical and functional correlations. Semin Arthritis Rheum, 2011. 40(5): p. 455-60. [2]Marot, M., et al., Prevalence and predictive factors of osteoporosis in systemic sclerosis patients: a case-control study. Oncotarget, 2015. 6(17): p. 14865-73. Disclosure of Interests Frederik Cosedis Enevoldsen: None declared, Josephine Therkildsen: None declared, Rasmus Klose-Jensen: None declared, Katja Thorup Aaen: None declared, Amanda Lynggaard Riis: None declared, Klaus Soendergaard: None declared, Ellen-Margrethe Hauge Grant/research support from: Personal fees from MSD, Pfizer, UCB, and Sobi. Grants from Roche and Novartis.

Background Prediction of the pattern of organ involvement in patients with diffuse cutaneous systemic sclerosis (dcSSc) is supported by the specificity of autoantibodies. These autoantibodies are generally mutually exclusive and highly specific for dcSSc 1 . The autoantibody reactivity allows patients to be stratified early in the disease course, leading to a tailored approach and management. Anti-topoisomerase I antibodies (ATA) are predictors of the development of interstitial lung disease (ILD) and digital ulcers but appear to be protective against pulmonary artery hypertension (PAH). Objectives The aim of this project was to elucidate the functioning of ATA and their role in the pathogenicity of the disease. Methods Sera from healthy volunteers (HV) and dcSSc patients with and without ATA were collected from Aarhus University Hospital. Total IgG was purified from the sera using a Protein G column. ATA was further purified from the total IgG fraction by passing it through a column packed with the functional domains of human topoisomerase-1 (TOP1) expressed from E.coli. Sera, total IgG and purified ATA fractions were utilized in preliminary experiments. The ability of ATA to inhibit TOP1 was tested by a in house developed REEAD assay in which the generation of closed DNA circles, by the cleavage-ligation activity of TOP1, is monitored after rolling circle amplification 2 . Results Serum from patients with dcSSc, but not from HV, could inhibit TOP1 activity at various dilutions. Similarly, purified total IgG fractions from dcSSc patients could also inhibit TOP1 activity. ATA was successfully separated from IgG from dcSSc patients who were positive for ATA. This could inhibit TOP1 activity even at lower concentrations than total IgG. This was the case for both mitochondrial and nuclear TOP1 activity. Fully in line with presence of ATA, PBMC’s from dcSSc patients contained a lower TOP1 activity compared to HVs PBMCs. Conclusion To our knowledge we are the first group to be able to successfully separate ATA from total IgG from dcSSc patients. our unique in house developed REEAD assay clearly demonstrates the function of ATA and provides the opportunity to better understand References [1]Role of autoantibodies in the diagnosis and prognosis of interstitial lung disease in autoimmune rheumatic disorders. Masataka Kuwana, Albert Gil-Vila and Albert Selva-O’Callaghan, Ther Adv Musculoskel Dis 2021, Vol. 13: 1–17 DOI: 10.1177/ [2]Rolling circle amplification based detection of human topoisomerase I activity on magnetic beads. Zuccaro, Laura; Tesauro, Cinzia; Cerroni, Barbara; Ottaviani, Alessio; Knudsen, Birgitta Ruth; Balasubramanian, Kannan; Desideri, Alessandro. Analytical Biochemistry, Bind 451, 15.04.2014, s. 42-44. Disclosure of Interests None declared

Gastrointestinal (GI) problems are common in patients with systemic sclerosis (SSc) and some develop pronounced underweight despite dietary intervention. Two patients with SSc were referred to our department because of severe GI symptoms and critical underweight. They had lost 24 and 23 kg, respectively, and their body mass index (BMI) was only 15 and 17 kg/m(2), respectively. After careful GI evaluation, both were treated with percutaneous endoscopic gastrostomy (PEG). Eight months later one patient had gained 13 kg (BMI 19 kg/m(2)) and after 5 months follow-up the other had gained 9 kg (BMI 20 kg/m(2)). Weight was no longer critically low and quality of life improved in both. In conclusion, PEG is an option in selected patients with severe malnutrition due to SSc. However, we advocate that thorough GI evaluation is performed in advance.