Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells

Summary We previously demonstrated a correlation between the frequency of CX3CR1-expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1(high) NK cells were more cytotoxic than their CX3CR1(neg/low) counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1(high) , CX3CR1(neg/low) NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin-2 and promoted a strong up-regulation of the key co-stimulatory molecule CD40 on monocytes. Co-expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1(neg)  CD56(bright) , CX3CR1(neg)  CD56(dim) and CX3CR1(high)  CD56(dim) fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56(dim) discriminated between an intermediary CX3CR1(neg)  CD56(dim) and fully mature CX3CR1(high)  CD56(dim) NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system

POS0744 A NEGATIVE INTERFERON BIOMARKER CD169 / SIGLEC-1 RULES OUT SYSTEMIC LUPUS ERYTHEMATOSUS

Background:While there have been advances in the therapy of systemic lupus erythematosus (SLE) in recent years, there have been no major new findings in SLE biomarkers [1, 2]. Type I interferon (IFN) plays a pivotal role in the pathogenesis of SLE [3]. In 2008, we first described CD169 / SIGLEC-1 (sialic acid-binding immunoglobulin-like lectin-1), an interferon-induced adhesion molecule on monocytes in SLE patients [4]. For over five years SIGLEC-1 has been routinely assessed in our clinic.Objectives:To evaluate and compare the diagnostic utility of the type I IFN induced SIGLEC-1 with established biomarkers in the initial diagnosis of the disease.Methods:We analyzed retrospectively 232 patients who were on suspicion of SLE at Charité University Hospital Berlin between October 2015 and September 2020. Patients underwent full clinical characterization, and biomarkers were determined in the routine laboratory. Based on the final diagnosis, we divided patients into two groups: A) initial diagnosis of SLE and B) Non-SLE mimicking condition.Results:In 76 patients (32.3 %) SLE was confirmed by fulfilling the EULAR / ACR 2019 classification criteria [5]. SIGLEC-1 was dramatically increased in patients with an initial diagnosis of SLE compared to patients without SLE (p&lt;0.0001). For a threshold of 2500 molecule per monocyte, a sensitivity of 98.7 %, a specificity of 82.1 %, a negative predictive value (NPV) of 99.2 %, and a positive predictive value (PPV) of 72.8 % were calculated for SIGLEC-1. Adjusted to the prevalence of SLE in Germany (36.7 per 100,000 inhabitants [6]) NPV and PPV turned out to &gt; 99.9 % and 0.2 %. We further aimed to compare not only the performance of the tests at a given cutoff but also across all possible measured values. Therefore, we conducted ROC curves analyses (see figure 1). The area under the curve (AUC) of SIGLEC-1 test was significantly higher than that of ANA test (AUC=0.88, p=0.031), C3 (AUC = 0.83, p=0.001), C4 (AUC=0.83, p=0.002), but not than that of the Anti-dsDNA ELISA (AUC=0.90, p=0.163).Conclusion:Our study shows that IFN activity is a hallmark at the onset of the disease and that the interferon biomarker SIGLEC-1 is valuable to rule out SLE in suspected cases.References:[1]Ostendorf L, Burns M, Durek P, Heinz GA, Heinrich F, Garantziotis P, Enghard P, Richter U, Biesen R, Schneider U et al: Targeting CD38 with Daratumumab in Refractory Systemic Lupus Erythematosus. N Engl J Med 2020, 383(12):1149-1155.[2]Furie R, Rovin BH, Houssiau F, Malvar A, Teng YKO, Contreras G, Amoura Z, Yu X, Mok CC, Santiago MB et al: Two-Year, Randomized, Controlled Trial of Belimumab in Lupus Nephritis. N Engl J Med 2020, 383(12):1117-1128.[3]Ronnblom L, Leonard D: Interferon pathway in SLE: one key to unlocking the mystery of the disease. Lupus Sci Med 2019, 6(1):e000270.[4]Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner M, Backhaus M, Haupl T, Rudwaleit M, Riemekasten G, Radbruch A et al: Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus. Arthritis Rheum 2008, 58(4):1136-1145.[5]Aringer M, Costenbader K, Daikh D, Brinks R, Mosca M, Ramsey-Goldman R, Smolen JS, Wofsy D, Boumpas DT, Kamen DL et al: 2019 European League Against Rheumatism/American College of Rheumatology classification criteria for systemic lupus erythematosus. Annals of the Rheumatic Diseases 2019, 78(9):1151-1159.[6]Brinks R, Fischer-Betz R, Sander O, Richter JG, Chehab G, Schneider M: Age-specific prevalence of diagnosed systemic lupus erythematosus in Germany 2002 and projection to 2030. Lupus 2014, 23(13):1407-1411.Disclosure of Interests:None declared</jats:sec

POS0183 SIGLEC1 AS A TYPE I INTERFERON BIOMARKER IN IDIOPATHIC INFLAMMATORY MYOPATHIES

Background:Idiopathic inflammatory myopathies (IIM) are autoimmune diseases that mainly affect skeletal muscle, lung, skin and joints. IIM can be separated into dermatomyositis (DM), inclusion body myositis (IBM), antisynthetase syndrome (AS) and immune-mediated necrotizing myopathy (IMNM). Type I interferons (IFN) are known to play a crucial role in the etiopathogenesis of some of these entities such as DM.[1] Sialic acid binding Ig-like lectin 1 (SIGLEC1, CD169) is part of the type I IFN signature found in SLE and DM and is expressed on the cell surface of monocytes. Thus, analysis of SIGLEC1 expression by flow cytometry enables a straightforward assessment of the type I IFN signature. Its utility has been shown for juvenile and adult SLE and other rheumatic diseases but not in IIM.[2,3] The assessment of the type I IFN system in clinical practice is an unmet need and, in this context, SIGLEC1 might be useful.Objectives:To assess SIGLEC1 expression on monocytes by flow cytometry as a type I IFN biomarker in IIMMethods:Pediatric and adult patients with a clinical diagnosis of DM, AS, IMNM and IBM and at least one measurement of SIGLEC1 who have been treated at the Department of Rheumatology, Charité - Universitätsmedizin Berlin between 2015 and 2020 were included in this retrospective study. Control groups of healthy individuals (n=19) and SLE patients (n=30) were included. Disease activity was assessed by Physician Global Assessment (PGA) and Childhood Myositis Assessment Scale (CMAS). SIGLEC1 expression on monocytes was analyzed by flow cytometry. Cross-sectional analyses (n=74) were performed using Mann Whitney-U test (MWU) and two-level mixed-effects linear regression model was used for longitudinal analyses (n=26, 110 visits). This study was approved by the local ethics committee of the Charité - Universitätsmedizin Berlin.Results:74 patients (adult/juvenile DM: n=21/n=17; AS: n=19; IMNM: n=8; IBM: n=9) were included. In cross-sectional analysis, SIGLEC1 expression was significantly upregulated in adult and juvenile DM patients with moderate to severe disease activity (PGA≥5) compared with adult/juvenile DM patients with no to moderate disease activity (PGA&lt;5) (both p&lt;0.001). In longitudinal analyses, SIGLEC1 correlated with disease activity in juvenile DM (SIGLEC1 vs. CMAS: betaST=-0.65; p&lt;0.001) and adult DM (SIGLEC1 vs. PGA: betaST=0.52; p&lt;0.001), better than Creatine Kinase (CK) (juvenile DM, CK vs. CMAS: betaST=-0.50; p&lt;0.001; adult DM, CK vs PGA: betaST=0.17; p=0.149). In AS 42,1% of the patients showed elevated SIGLEC1 expression, while it was not upregulated in IMNM and only in two patients with IBM, who were concurrently positive for autoantibodies that affect the type I IFN system (see Figure 1).Conclusion:SIGLEC1 is a useful biomarker to identify an activated type I IFN system in IIM. Flow cytometry is used widely in laboratory medicine, which could facilitate the implementation of SIGLEC1 into clinical routine.References:[1]Gallay L, Mouchiroud G, Chazaud B. Interferon-signature in idiopathic inflammatory myopathies: Current Opinion in Rheumatology 2019;31:634–42. doi:10.1097/BOR.0000000000000653[2]Rose T, Grutzkau A, Hirseland H, et al. IFNalpha and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus. Ann Rheum Dis 2013;72:1639–45. doi:10.1136/annrheumdis-2012-201586[3]Stuckrad SL von, Klotsche J, Biesen R, et al. SIGLEC1 (CD169) is a sensitive biomarker for the deterioration of the clinical course in childhood systemic lupus erythematosus. Lupus 2020;:961203320965699. doi:10.1177/0961203320965699Figure 1.SIGLEC1 expression on monocytes in IIM subgroups and control groups; in IIM subgroups, patients with low disease activity (PGA&lt;5) are marked in blue, patients with high disease activity (PGA≥5) are marked in red; mAb/cell, monoclonal antibodies bound per cellDisclosure of Interests:None declared</jats:sec