Knockdown of SIGLEC1 inhibits osteogenic differentiation to alleviate ankylosing spondylitis progression by suppressing the TGF-ß1/SMAD signaling pathway.

Ankylosing spondylitis (AS) is a chronic inflammatory joint disease, which can result in disability in severe cases and endanger physical health. Two microarray datasets related to AS were selected from NCBI for bioinformatics analysis. Differentially expressed genes (DEGs) were screened and protein-protein interaction network was constructed to obtain hub genes. hSMSCs were injected with TNF-α to construct AS cell models. The hSMSCs were transfected with SIGLEC1 siRNA to silence SIGLEC1 expression. CCK-8, western blot, qRT-PCR, and ELISA were performed to detect the effects of SIGLEC1 knockdown on cell proliferation, osteogenic differentiation (ALP, BMP2, Osterix, and Runx2), inflammation (IL-23 and IL-6), and TGF-ß1/SMAD signaling pathway (SMAD3, SMAD7 and TGF-ß1). A TGF-ß1 activator was applied for feedback function assays. A total of 29 common DEGs were screened from GSE181364 and GSE221786, and the key gene SIGLEC1 was selected. Knockdown of SIGLEC1 promoted cell proliferation and inhibited ALP activity, the level of BMP2, Osterix and Runx2 in TNF-α-induced hSMSCs, as well as the decreased inflammatory factors IL-23 and IL-6. Furthermore, knockdown of SIGLEC1 inhibited the expression of TGF-ß1/SMAD signaling pathway related proteins, while the treatment of TGF-ß1 activator weakened the inhibiting effects of sh-SIGLEC1 on the osteogenic differentiation and inflammation in TNF-α-induced hSMSCs. In summary, knockdown of SIGLEC1 promoted cell proliferation and inhibited osteogenic differentiation and inflammation by inhibiting TGF-ß1/SMAD signaling pathway, thereby suppressing the development of AS.

Intra-patient spatial comparison of non-metastatic and metastatic lymph nodes reveals the reduction of CD169+ macrophages by metastatic breast cancers.

BACKGROUND: Breast cancer cells suppress the host immune system to efficiently invade the lymph nodes; however, the underlying mechanism remains incompletely understood. Here, we aimed to comprehensively characterise the effects of breast cancers on immune cells in the lymph nodes. METHODS: We collected non-metastatic and metastatic lymph node samples from 6 patients with breast cancer with lymph node metastasis. We performed bulk transcriptomics, spatial transcriptomics, and imaging mass cytometry to analyse the obtained lymph nodes. Furthermore, we conducted histological analyses against a larger patient cohort (474 slices from 58 patients). FINDINGS: The comparison between paired lymph nodes with and without metastasis from the same patients demonstrated that the number of CD169+ lymph node sinus macrophages, an initiator of anti-cancer immunity, was reduced in metastatic lymph nodes (36.7 ± 21.1 vs 7.3 ± 7.0 cells/mm2, p = 0.0087), whereas the numbers of other major immune cell types were unaltered. We also detected that the infiltration of CD169+ macrophages into metastasised cancer tissues differed by section location within tumours, suggesting that CD169+ macrophages were gradually decreased after anti-cancer reactions. Furthermore, CD169+ macrophage elimination was prevalent in major breast cancer subtypes and correlated with breast cancer staging (p = 0.022). INTERPRETATION: We concluded that lymph nodes with breast cancer metastases have fewer CD169+ macrophages, which may be detrimental to the activity of anti-cancer immunity. FUNDING: JSPS KAKENHI (16H06279, 20H03451, 20H04842, 22H04925, 19K16770, and 21K15530, 24K02236), JSPS Fellows (JP22KJ1822), AMED (JP21ck0106698), JST FOREST (JPMJFR2062), Caravel, Co., Ltd, Japan Foundation for Applied Enzymology, and Sumitomo Pharma Co., Ltd. under SKIPS.

Serum and urine interferon-inducible protein 10, galectin-9, and SIGLEC-1 as biomarkers of disease activity in systemic lupus erythematosus.

Background/aim: In this prospective study, we aimed to investigate the association of serum (s) and urine (u) IP-10, galectin-9, and SIGLEC-1 with disease activity in patients with systemic lupus erythematosus (SLE). Materials and methods: Sixty-three patients with active SLE (31 renal, 32 extrarenal) were included. Thirty patients with inactive SLE (15 renal, 15 extrarenal), 17 with renal active AAV, and 32 healthy volunteers were selected as control groups. Serum and urine IP-10, galectin-9, and SIGLEC-1 were tested using ELISA. Results: Levels of sIP-10 (p = 0.046), uIP-10 (p < 0.001), sGalectin-9 (p = 0.03), and uSIGLEC-1 (p = 0.006) were significantly higher in active SLE group compared to the inactive SLE; however, no differences were detected in the comparison of uGalectin-9 (p = 0.18) and sSIGLEC-1 (p = 0.69) between two groups. None of the biomarkers discriminated patients with active renal SLE from active extrarenal SLE. ROC analyses revealed an AUC of 0.63 (0.52-0.73) for sIP-10, 0.78 (0.68-0.86) for uIP-10, 0.64 (0.53-0.74) for sGalectin-9, and 0.68 (0.57-0.77) for uSIGLEC-1 in discriminating disease activity in SLE, which did not outperform C3 (0.75, 0.64-0.84) and C4 (0.72, 0.61-0.82). sIP-10 (p = 0.001), uIP-10 (p = 0.042), and uGalectin-9 (p = 0.009) were significantly increased in patients with active renal SLE compared to active renal AAV. sGalectin-9 (p < 0.001) and sIP-10 levels (p = 0.06) were decreased after 8 (5-22.5) months of treatment. Conclusion: sIP-10, uIP-10, sGalectin-9, and uSIGLEC-1 reflect global disease activity in SLE but do not outperform C3 and C4. sIP-10 and uIP-10 may be specific for active SLE compared to active AAV. sIP-10 and sGalectin-9 might be valuable in monitoring response after treatment.

Siglec-1, an easy and contributory inflammation marker in rheumatology.

Objectives: Inflammatory markers such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are poorly informative about interferon (IFN)-related disorders. In these conditions, the measure of the interferon score (IS), obtained by measuring the expression of IFN-stimulated genes, has been proposed. Flow cytometry-based assays measuring sialic-acid-binding Ig-like lectin 1 (Siglec-1) expression could be a more practical tool for evaluating IFN-inflammation. The study compared Siglec-1 measures with IS and other inflammatory indexes. We compared Siglec-1 measures with IS and other inflammatory indexes in real-world paediatric rheumatology experience. Methods: We recruited patients with immuno-rheumatological conditions, acute infectious illness and patients undergoing orthopaedic surgery as controls. Siglec-1 expression was measured in all samples, and IS, ESR and CRP were also recorded if available. Results: Overall, 98 subjects were enrolled in the study, with a total of 104 measures of Siglec-1. Compared with IS, Siglec-1 expression showed good accuracy (86.0%), specificity (72.7%) and sensitivity (85.7%). The measure of the percentage of Siglec-1-positive cells performed best at low levels of IFN-inflammation, while the measure of mean fluorescence intensity performed best at higher levels. Ex vivo studies on IFN-stimulated monocytes confirmed this behaviour. There was no link between Siglec-1 expression and either ESR or CRP, and positive Siglec-1 results were found even when ESR and CRP were normal. A high Siglec-1 expression was also recorded in subjects with acute infections. Conclusion: Siglec-1 measurement by flow cytometry is an easy tool to detect IFN-related inflammation, even in subjects with normal results of common inflammation indexes.

Identification of Siglec-1-negative alveolar macrophages with proinflammatory phenotypes in chronic obstructive pulmonary disease.

Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. However, subpopulations of AMs participating in chronic inflammation have been poorly characterized. We previously reported that Siglec-1 expression on AMs, which is important for bacteria engulfment, was decreased in COPD. Here, we show that Siglec-1-negative AMs isolated from COPD lung tissues exhibit a proinflammatory phenotype and are associated with poor clinical outcomes in patients with COPD. Using flow cytometry, we segregated three subsets of AMs based on the expression of Siglec-1 and their side scattergram (SSC) and forward scattergram (FSC) properties: Siglec-1+SSChiFSChi, Siglec-1-SSChiFSChi, and Siglec-1-SSCloFSClo subsets. The Siglec-1-SSCloFSClo subset number was increased in COPD. RNA sequencing revealed upregulation of multiple proinflammatory signaling pathways and emphysema-associated matrix metalloproteases in the Siglec-1-SSCloFSClo subset. Gene set enrichment analysis indicated that the Siglec-1-SSCloFSClo subset adopted intermediate phenotypes between monocytes and mature alveolar macrophages. Functionally, these cells produced TNF-α, IL-6, and IL-8 at baseline, and these cytokines were significantly increased in response to viral RNA. The increase in Siglec-1-negative AMs in induced sputum is associated with future exacerbation risk and lung function decline in patients with COPD. Collectively, the novel Siglec-1-SSCloFSClo subset of AMs displays proinflammatory properties, and their emergence in COPD airways may be associated with poor clinical outcomes.NEW & NOTEWORTHY Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. We find that Siglec-1-negative alveolar macrophages have a wide range of proinflammatory landscapes and a protease-expressing phenotype. Moreover, this subset is associated with the pathogenesis of COPD and responds to viral stimuli.

A novel subpopulation of monocytes with a strong interferon signature indicated by SIGLEC-1 is present in patients with in recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes is a T cell-mediated autoimmune disease characterised by pancreatic beta cell destruction. In this study, we explored the pathogenic immune responses in initiation of type 1 diabetes and new immunological targets for type 1 diabetes prevention and treatment. METHODS: We obtained peripheral blood samples from four individuals with newly diagnosed latent autoimmune diabetes in adults (LADA) and from four healthy control participants. Single-cell RNA-sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells to uncover transcriptomic profiles of early LADA. Validation was performed through flow cytometry in a cohort comprising 54 LADA, 17 adult-onset type 2 diabetes, and 26 healthy adults, matched using propensity score matching (PSM) based on age and sex. A similar PSM method matched 15 paediatric type 1 diabetes patients with 15 healthy children. Further flow cytometry analysis was performed in both peripheral blood and pancreatic tissues of non-obese diabetic (NOD) mice. Additionally, cell adoptive transfer and clearance assays were performed in NOD mice to explore the role of this monocyte subset in islet inflammation and onset of type 1 diabetes. RESULTS: The scRNA-seq data showed that upregulated genes in peripheral T cells and monocytes from early-onset LADA patients were primarily enriched in the IFN signalling pathway. A new cluster of classical monocytes (cluster 4) was identified, and the proportion of this cluster was significantly increased in individuals with LADA compared with healthy control individuals (11.93% vs 5.93%, p=0.017) and that exhibited a strong IFN signature marked by SIGLEC-1 (encoding sialoadhesin). These SIGLEC-1+ monocytes expressed high levels of genes encoding C-C chemokine receptors 1 or 2, as well as genes for chemoattractants for T cells and natural killer cells. They also showed relatively low levels of genes for co-stimulatory and HLA molecules. Flow cytometry analysis verified the elevated levels of SIGLEC-1+ monocytes in the peripheral blood of participants with LADA and paediatric type 1 diabetes compared with healthy control participants and those with type 2 diabetes. Interestingly, the proportion of SIGLEC-1+ monocytes positively correlated with disease activity and negatively with disease duration in the LADA patients. In NOD mice, the proportion of SIGLEC-1+ monocytes in the peripheral blood was highest at the age of 6 weeks (16.88%), while the peak occurred at 12 weeks in pancreatic tissues (23.65%). Adoptive transfer experiments revealed a significant acceleration in diabetes onset in the SIGLEC-1+ group compared with the SIGLEC-1- or saline control group. CONCLUSIONS/INTERPRETATION: Our study identified a novel group of SIGLEC-1+ monocytes that may serve as an important indicator for early diagnosis, activity assessment and monitoring of therapeutic efficacy in type 1 diabetes, and may also be a novel target for preventing and treating type 1 diabetes. DATA AVAILABILITY: RNA-seq data have been deposited in the GSA human database ( https://ngdc.cncb.ac.cn/gsa-human/ ) under accession number HRA003649.

CD169+ macrophage intrinsic IL-10 production regulates immune homeostasis during sepsis.

Macrophages facilitate critical functions in regulating pathogen clearance and immune homeostasis in tissues. The remarkable functional diversity exhibited by macrophage subsets is dependent on tissue environment and the nature of the pathological insult. Our current knowledge of the mechanisms that regulate the multifaceted counter-inflammatory responses mediated by macrophages remains incomplete. Here, we report that CD169+ macrophage subsets are necessary for protection under excessive inflammatory conditions. We show that in the absence of these macrophages, even under mild septic conditions, mice fail to survive and exhibit increased production of inflammatory cytokines. Mechanistically, CD169+ macrophages control inflammatory responses via interleukin-10 (IL-10), as CD169+ macrophage-specific deletion of IL-10 was lethal during septic conditions, and recombinant IL-10 treatment reduced lipopolysaccharide (LPS)-induced lethality in mice lacking CD169+ macrophages. Collectively, our findings show a pivotal homeostatic role for CD169+ macrophages and suggest they may serve as an important target for therapy under damaging inflammatory conditions.

Impact of Siglecs on autoimmune diseases.

Autoimmune diseases affect tens of millions of people just in the United States alone. Most of the available treatment options are aimed at reducing symptoms but do not lead to cures. Individuals affected with autoimmune diseases suffer from the imbalance between tolerogenic and immunogenic functions of their immune system. Often pathogenesis is mediated by autoreactive B and T cells that escape central tolerance and react against self-antigens attacking healthy tissues in the body. In recent years Siglecs, sialic-acid-binding immunoglobulin (Ig)-like lectins, have gained attention as immune checkpoints for therapeutic interventions to dampen excessive immune responses and to restore immune tolerance in autoimmune diseases. Many Siglecs function as inhibitory receptors suppressing activation signals in various immune cells through binding to sialic acid ligands as signatures of self. In this review, we highlight potential of Siglecs in suppressing immune responses causing autoimmune diseases. In particular, we cover the roles of CD22 and Siglec-G/Siglec-10 in regulating autoreactive B cell responses. We discuss several functions of Siglec-10 in the immune modulation of other immune cells, and the potential of therapeutic strategies for restoring immune tolerance by targeting Siglecs and expanding regulatory T cells. Finally, we briefly review efforts evaluating Siglec-based biomarkers to monitor autoimmune diseases.

Adaptation of African swine fever virus to porcine kidney cells stably expressing CD163 and Siglec1.

African swine fever virus (ASFV) is a complex large DNA enveloped virus that causes African swine fever (ASF) with a fatality rate of up to 100%, seriously threatening the global swine industry. Due to the strict cell tropism of ASFV, there is no effective in vitro cell line, which hinders its prevention and control. Herein, we analyzed genome-wide transcriptional profiles of ASFV-susceptible porcine alveolar macrophages (PAMs) and non-susceptible cell lines PK15 and 3D4-21, an found that PAM surface pattern recognition receptors (PRRs) were significantly higher and common differential genes were significantly enriched in phagocytosis compared with that observed in PK15 and 3D4-21 cell lines. Therefore, endocytosis functions of host cell surface PRRs may play key roles in ASFV infection in vitro. ASFV was found to be infective to PK15 and 3D4-21 cell lines overexpressing CD163 and Siglec1, and to the PK15S1-CD163 cell line stably expressing CD163 and Siglec1. However, the PK15 and 3D4-21 cell lines overexpressing CD163 or Siglec1 alone were not infectious. Simultaneous interference of CD163 and Siglec1 in PAMs with small interfering RNA (siRNA) significantly reduced the infectivity of ASFV. However, siRNA interference of CD163 and Siglec1 respectively did not affect ASFV infectivity. ASFV significantly inhibited IFN expression levels in PAMs and PK15S1-CD163 cells, but had no effect on PK15 and 3D4-21 cell lines. These results indicate that CD163 and Siglec1 are key receptors for ASFV-infected host cells, and both play a synergistic role in the process of ASFV infection. ASFV inhibits IFN expression in susceptible cells, thereby downregulating the host immune response and evading the immune mechanism. The discovery of the ASFV receptor provides novel ideas to study ASFV and host cell interactions, pathogenic mechanisms, development of receptor blockers, vaccine design, and disease resistance breeding.

Myeloid CD169/Siglec1: An immunoregulatory biomarker in viral disease.

CD169, also known as Siglec1 or Sialoadhesin (Sn), is a surface adhesion molecule on human myeloid cells. Being part of the Siglec family, it acts as a receptor for sialylated molecular structures, which are found among various pathogenic and non-pathogenic ligands. Recent data suggest that CD169 may represent a promising new biomarker in acute respiratory and non-respiratory viral infections, such as SARS-CoV-2, Respiratory syncytial virus (RSV) and Human immunodeficiency virus (HIV). Therein lies a great potential to sufficiently differentiate viral from bacterial infection, which has been an incessant challenge in the clinical management of infectious disease. CD169 equips myeloid cells with functions, reaching far beyond pathogen elimination. In fact, CD169 seems to crosslink innate and adaptive immunity by antigen presentation and consecutive pathogen elimination, embodying a substantial pillar of immunoregulation. Yet, our knowledge about the kinetics, mechanisms of induction, signaling pathways and its precise role in host-pathogen interaction remains largely obscure. In this review, we describe the role of CD169 as a potentially novel diagnostic biomarker for respiratory viral infection by evaluating its strengths and weaknesses and considering host factors that are involved in pathogenesis of virus infection. Finally, this brief review aims to point out shortcomings of available evidence, thus, guiding future work revolving the topic.

SIGLEC-1 in Systemic Sclerosis: A Useful Biomarker for Differential Diagnosis.

Systemic Sclerosis (SSc) is a clinically heterogeneous disease that includes an upregulation of type I interferons (IFNs). The aim of this observational study was to investigate the IFN-regulated protein Sialic Acid−Binding Ig-like Lectin 1 (SIGLEC-1) as a biomarker for disease phenotype, therapeutic response, and differential diagnosis in SSc. Levels of SIGLEC-1 expression on monocytes of 203 SSc patients were determined in a cross-sectional and longitudinal analysis using multicolor flow cytometry, then compared to 119 patients with other rheumatic diseases and 13 healthy controls. SSc patients higher SIGLEC-1 expression on monocytes (2097.94 ± 2134.39) than HCs (1167.45 ± 380.93; p = 0.49), but significantly lower levels than SLE (8761.66 ± 8325.74; p < 0.001) and MCTD (6414.50 ± 1846.55; p < 0.001) patients. A positive SIGELC-1 signature was associated with reduced forced expiratory volume (p = 0.007); however, we were unable to find an association with fibrotic or vascular disease manifestations. SIGLEC-1 remained stable over time and was independent of changes in immunosuppressive therapy. However, SIGLEC-1 is suitable for differentiating SSc from other connective tissue diseases. SIGLEC-1 expression on monocytes can be useful in the differential diagnosis of connective tissue disease but not as a biomarker for SSc disease manifestations or activity.

Ganglioside-Functionalized Nanoparticles for Chimeric Antigen Receptor T-Cell Activation at the Immunological Synapse.

Chimeric Antigen Receptor (CAR) T cell therapy has proven to be an effective strategy against hematological malignancies but persistence and activity against solid tumors must be further improved. One emerging strategy for enhancing efficacy is based on directing CAR T cells to antigen presenting cells (APCs). Activation of CAR T cells at the immunological synapse (IS) formed between APC and T cell is thought to promote strong, persistent antigen-specific T cell-mediated immune responses but requires integration of CAR ligands into the APC/T-cell interface. Here, we demonstrate that CAR ligand functionalized, lipid-coated, biodegradable polymer nanoparticles (NPs) that contain the ganglioside GM3 (GM3-NPs) bind to CD169 (Siglec-1)-expressing APCs and localize to the cell contact site between APCs and CAR T cells upon initiation of cell conjugates. The CD169+ APC/CAR T-cell interface is characterized by a strong optical colocalization of GM3-NPs and CARs, enrichment of F-actin, and recruitment of ZAP-70, indicative of integration of GM3-NPs into a functional IS. Ligands associated with GM3-NPs localized to the APC/T-cell contact site remain accessible to CARs and result in robust T-cell activation. Overall, this work identifies GM3-NPs as a potential antigen delivery platform for active targeting of CD169 expressing APCs and enhancement of CAR T-cell activation at the NP-containing IS.

The role of activated monocyte IFN/SIGLEC1 signalling in Graves' disease.

Graves' disease (GD) is characterized by dysregulation of the immune system with aberrant immune cell function. However, there have been few previous studies on the role of monocytes in the pathology of GD. The object of this study was to investigate whether and how monocytes participate in GD pathology. CD14+ monocytes were isolated from untreated initial GD patients and healthy controls. Then, RNA-seq was performed to investigate changes in global mRNA expression in monocytes and found that type I interferon (IFN) signalling was among the top upregulated signalling pathways in GD monocytes. Type I IFN-induced sialic acid-binding immunoglobulin-like lectin1 (SIGLEC1) expression was significantly upregulated in untreated GD patients and correlated with thyroid parameters. Patient serum SIGLEC1 concentrations were reduced after anti-thyroid drug treatment. Inhibiting SIGLEC1 expression could inhibit proinflammatory cytokine (IL-1ß, IL-6, IL-8, IL-10 and M-CSF) expression in monocytes. In conclusion, our study suggested that type I IFN-mediated monocyte activation could have a deleterious effect on the pathogenesis of GD. These observations indicated that the inhibition of type I IFN-activated monocytes/macrophages could have a therapeutic effect on GD remission.

A Microbiota-Dependent Subset of Skin Macrophages Protects Against Cutaneous Bacterial Infection.

Microbiota is essential to the development and functional maturation of the immune system. The effects of the gut microbiota on myeloid cells remote from the gut, especially the skin remain unclear. Transcriptomic analysis revealed that type I interferon (IFN) signaling was down-regulated in the skin of germ-free mice compared to that in specific pathogen-free mice. The decrease in type I IFN signaling was closely related to the presence of microbiota and macrophage-specific marker CD169. The absence of CD169+ macrophages resulted in increased bacterial burden and impaired immune responses against Staphylococcus aureus skin infection. CD169+ macrophages mediated the recruitment of γδ T cells as well as the activation of γδ T cells via interleukin (IL)-23. Our findings demonstrate the role of the microbiota in establishment of a specific myeloid cell subset expressing CD169 in the skin and provide evidence of a specific mechanism by which this subset protects against bacterial skin infection.

Type I IFNs repolarized a CD169+ macrophage population with anti-tumor potentials in hepatocellular carcinoma.

Macrophages constitute a major component in human hepatocellular carcinoma (HCC) and perform various functions to facilitate disease progression. Reprogramming or reconstituting the tumor surveillance phenotypes of macrophages represents an attractive immunotherapeutic strategy in cancer treatments. The current study identified CD169 as a potential target for macrophage repolarization since it signified a population of macrophages positively correlated with an activated immune signature and better prognosis of patients with HCC. In vitro experiments revealed that a low dose of type I interferon (IFN) could effectively reprogram human monocyte-derived macrophages to upregulate CD169 expression, and such induced CD169+ macrophages exhibited significantly enhanced phagocytotic and CD8+ T cell-activating capacities compared to controls. A low dose of IFNα also inhibited hepatoma growth in mice in vivo, presumably through polarizing the CD169+ macrophage population and enhancing CD8+ T cell activities. Notably, IFNα also induced substantial PD-L1 expression on macrophages in vivo, and thus blockade of PD-L1 could further increase the anti-tumor efficacy of IFNα in the treatment of HCC. We propose a low dose of IFNα in combination with a PD-L1 blocking agent as a potential anti-tumor therapeutic strategy via its effects on macrophage polarization.

Metabolic installation of macrophage-recruiting glycan ligand on tumor cell surface for in vivo tumor suppression.

Synthetic probes that could direct immune cells against tumors are potential immunotherapeutics. We herein report in vivo tumor suppression via an intravenously injected abiotic sialic acid (TCCSia) that could be metabolically incorporated into tumor cell surface to yield of a high affinity ligand (TCCSiaα2,3-Gal) of Siglec-1 specifically expressed on macrophages. We observed marked suppression of pulmonary metastasis and subcutaneous tumor growth of B16F10 melanoma cells in mice with TCCSia, suggesting the utility of abiotic sialic acid to modulate tumor immunity via recruiting Siglec+ immune cells.

Evaluation of SIGLEC1 in the diagnosis of suspected systemic lupus erythematosus.

OBJECTIVES: To evaluate and compare the diagnostic accuracy of SIGLEC1, a surrogate marker of type I IFN, with established biomarkers in an inception cohort of systemic lupus erythematosus (SLE). METHODS: SIGLEC1 was analysed by flow cytometry in 232 patients referred to our institution with suspected SLE between October 2015 and September 2020. RESULTS: SLE was confirmed in 76 of 232 patients (32.8 %) according to the 2019 EULAR/ACR classification criteria and their SIGLEC1 values were significantly higher compared with patients without SLE (P <0.0001). 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 SIGLEC1. Adjusted to the highest reported prevalence of SLE, the NPV and PPV were >99.9 % and 0.1 %, respectively. Using receiver operating characteristic (ROC) analysis and DeLong testing, the area under the curve (AUC) for SIGLEC1 (AUC = 0.95) was significantly higher than for ANA (AUC = 0.88, P = 0.031), C3 (AUC = 0.83, P = 0.001) and C4 (AUC = 0.83, P = 0.002) but not for anti-dsDNA antibodies (AUC = 0.90, P = 0.163). CONCLUSION: IFN-I pathway activation is detectable in almost all newly diagnosed SLE patients. Thus, a negative test result for SIGLEC1 is powerful to exclude SLE in suspected cases.

Siglec-1 expression on monocytes is associated with the interferon signature in juvenile dermatomyositis and can predict treatment response.

OBJECTIVE: JDM is a rare chronic immune-mediated inflammatory disease with a predominant role for type I IFN responses. We aimed to determine the potential of Siglec-1 expression on monocytes as a novel IFN-inducible biomarker for disease activity monitoring and prediction of treatment response in patients with JDM. METHODS: Siglec-1 was measured by flow cytometry on circulating monocytes of 21 newly diagnosed JDM patients before start of treatment and, for 10 of these, also during follow-up. The expression levels of five type I IFN-stimulated genes, MX1, IFI44, IFI44L, LY6E and IFIT3, were measured by RT-qPCR to determine the IFN signature and calculate an IFN score. IFN-inducible plasma proteins CXCL10 and galectin-9 were measured by multiplex immunoassay. RESULTS: Siglec-1 and IFN score were increased in JDM patients compared with controls and correlated with clinical disease activity. Stratification of patients by Siglec-1 expression at diagnosis identified those with high Siglec-1 expression as having a higher risk of requiring treatment intensification within the first 3 months after diagnosis (55% vs 0% of patients, P = 0.01). Siglec-1 expression strongly correlated with plasma levels of previously validated biomarkers CXCL10 (rs = 0.81, P < 0.0001) and galectin-9 (rs = 0.83, P < 0.0001), and was superior to the IFN score in predicting treatment response (area under the curve 0.87 vs 0.53, P = 0.01). CONCLUSION: Siglec-1 on monocytes is a novel IFN-inducible biomarker in JDM that correlates with clinical disease activity and identifies patients at risk for a suboptimal treatment response. Further studies are required to validate these findings and their clinical potential.

Intrinsic Defects in B Cell Development and Differentiation, T Cell Exhaustion and Altered Unconventional T Cell Generation Characterize Human Adenosine Deaminase Type 2 Deficiency.

PURPOSE: Deficiency of adenosine deaminase type 2 (ADA2) (DADA2) is a rare inborn error of immunity caused by deleterious biallelic mutations in ADA2. Clinical manifestations are diverse, ranging from severe vasculopathy with lacunar strokes to immunodeficiency with viral infections, hypogammaglobulinemia and bone marrow failure. Limited data are available on the phenotype and function of leukocytes from DADA2 patients. The aim of this study was to perform in-depth immunophenotyping and functional analysis of the impact of DADA2 on human lymphocytes. METHODS: In-depth immunophenotyping and functional analyses were performed on ten patients with confirmed DADA2 and compared to heterozygous carriers of pathogenic ADA2 mutations and normal healthy controls. RESULTS: The median age of the patients was 10 years (mean 20.7 years, range 1-44 years). Four out of ten patients were on treatment with steroids and/or etanercept or other immunosuppressives. We confirmed a defect in terminal B cell differentiation in DADA2 and reveal a block in B cell development in the bone marrow at the pro-B to pre-B cell stage. We also show impaired differentiation of CD4+ and CD8+ memory T cells, accelerated exhaustion/senescence, and impaired survival and granzyme production by ADA2 deficient CD8+ T cells. Unconventional T cells (i.e. iNKT, MAIT, Vδ2+ γδT) were diminished whereas pro-inflammatory monocytes and CD56bright immature NK cells were increased. Expression of the IFN-induced lectin SIGLEC1 was increased on all monocyte subsets in DADA2 patients compared to healthy donors. Interestingly, the phenotype and function of lymphocytes from healthy heterozygous carriers were often intermediate to that of healthy donors and ADA2-deficient patients. CONCLUSION: Extended immunophenotyping in DADA2 patients shows a complex immunophenotype. Our findings provide insight into the cellular mechanisms underlying some of the complex and heterogenous clinical features of DADA2. More research is needed to design targeted therapy to prevent viral infections in these patients with excessive inflammation as the overarching phenotype.

CD169 Defines Activated CD14+ Monocytes With Enhanced CD8+ T Cell Activation Capacity.

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.