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Background Systemic sclerosis (SSc) is a chronic autoimmune disease affecting multiple organs. It is characterized by excessive extracellular matrix deposition in the skin and internal organs accompanied by vascular damage and alterations of immune function. Regulatory T cells take part in the regulation of the immune system, development of tolerance against self antigens and the inhibition of autoimmunity. The role of Tregs seem to be essential in the pathogenesis of SSc. Objectives The aim of our work was to study the changes in the ratio of Treg populations and their cytokine production in peripheral blood samples of SSc patients compared to healthy controls (HC) and to evaluate the changes in relation to the activity and severity of the disease. Methods Peripheral blood samples were collected from SSc patients regularly visiting the Department of Rheumatology and Immunology not on immunosuppressive therapy and from HCs. PBMCs were isolated using ficoll gradient centrifugation and stimulated with the combination of PMA/ionomycin/brefeldin. Tregs (CD4+CD25+Foxp3+CD127-), Treg subgroups based on CD62L positivity and their cytokine production (IL-10, TGFβ) were investigated using flow cytometry. Results The ratio of Treg cells was increased in SSc compared to HCs while percentage of the IL-10 producing Tregs was lower. The ratio of CD62L+ Treg cells was higher in SSc than in HCs and their TGFβ producing proportion was decreased. The percentage of the IL-10 producing CD62L+ Treg cells was lower in active SSc compared to the inactive group. Conclusions According to our results the alterations of the ratio of Treg populations and their cytokine producing proportion in SSc patients is different from HCs and correlates with the activity of the disease. Consequently our study can help the early diagnostics of SSc and the assessment of its activity. Disclosure of Interest None declared

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular injury, autoimmune phenomena, inflammation, and fibrosis of the skin and various internal organs. Several lines of evidence indicate that abnormal B-cell function plays a key role in the development of SSc. The anti-CD20 monoclonal antibody therapy seems to show some clinical efficacy in SSc further emphasizing the importance of B cells in the pathomechanism of the disease. The B-cell compartment in peripheral blood of SSc patients contains an elevated number of naive and a decreased number of memory B cells. Objectives The aim of the present research was to set up an algorithm for the extended analysis of these B-cell subsets and to evaluate the clinical significance of the defined subpopulations. Methods Peripheral blood samples were obtained from SSc patients and healthy controls, PBMCs were isolated using ficoll gradient centrifugation, followed by magnetic bead separation of CD19+ B cells. Multiparametric flow cytometry was performed with antibodies specific for CD27, IgD, CD80, CD95 molecules. Detection of CD27 and IgD was applied to distinguish between naive (CD27-IgD+) and memory (CD27+) B cells. IgD posivity was also used to separate non-switched (CD27+IgD+) and switched (CD27+IgD-) memory subsets. In addition to expression of CD80, which provides a co-stimulatory signal necessary for T cell activation and survival, expression of CD95 – FAS receptor was also examined to investigate the activation state of the previously identified B cell subpopulations. Results The ratio of naive B cells was higher, the proportion of memory B cells, including both subsets, was decreased in SSc patients compared to healthy controls. Among SSc patients the ratio of switched memory and CD95+ memory B cells was higher in diffuse cutaneous SSc and in patients with pulmonary fibrosis. The proportion of switched memory B cells was also elevated in the anti-Scl-70 antibody positive group compared to ACA positive patients. In dcSSc patients the ratio of CD95+ memory B cells was also higher. Conclusions According to our results detailed flow cytometric analysis of naive and memory B-cell subsets could contribute to better distinction between the two SSc subtypes and to the evaluation of disease severity, consequently may be a useful new tool in routine immunological diagnostics. Acknowledgements This work was supported by Hungarian Scientific Research Fund - OTKA 75912 and 112939. Disclosure of Interest None declared

Background Natural autoantibodies bind to evolutionarily conserved antigens in organs and tissues of the body and may have regulatory functions in the immune system and play a role in the clearance of apoptotic cells. It has been known that some patients treated with anti-TNF-α agents develop autoantibodies, including antinuclear and anti-double-stranded DNA antibodies. However, in patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) treated with TNF-α blockers there have been no studies regarding the presence of natural autoantibodies against citrate synthase (CS), a mitochondrial inner membrane enzyme, and DNA topoisomerase I (topo I), an enzyme that relaxes the superhelical stress in DNA. Objectives Our aim was: (1) to investigate whether patients with RA and SpA receiving anti-TNF-α treatment produce natural autoantibodies to CS and topo I; and (2) to test whether such therapy affects the levels of these autoantibodies. Methods The study included a total of 36 patients (20 with RA, 16 with SpA) who received certolizumab or etanercept therapy. The levels of anti-CS and anti-topo I (immunodominant fragment) autoantibodies (IgM and IgG) were measured in the blood serum using enzyme-linked immunosorbent assay developed in our laboratory. The measurements were done at the starting point (0), 6, or 12 months of therapy. Results In the combined patient group (RA + SpA) we detected significantly increased levels of anti-CS IgM and IgG antibodies after 6 and 12 months of anti-TNF-α therapy. The anti-topo I IgM and IgG were significantly elevated only after 12 months of therapy, when compared to baseline antibody levels before the start of treatment. Conclusions The significance of the presence of anti-CS and anti-topo I natural autoantibodies is still a subject of research. We hypothesize that the increase in the levels of these natural autoantibodies might be a marker for the functional role of the natural immune system in patients treated with anti-TNF-α blockers. Because TNF-α is a pleiotropic cytokine, blocking its function can elicit various side-effects, including hematological, immunological, and others. Natural autoantibodies may function in the clearance of apoptotic cells and they may help restore the immunological milieu by reducing inflammation and protecting against some infections, functions in which the natural immune system has been implicated. Disclosure of Interest None declared