Prophylactic antibodies inhibit spike-specific T and B cell responses after COVID-19 vaccination.

Active and passive immunization is used in high-risk patients to prevent severe courses of COVID-19, but the impact of prophylactic neutralizing antibodies on the immune reaction to the mRNA vaccines has remained enigmatic. Here we show that CD4 T and B cell responses to Spikevax booster immunization are suppressed by the therapeutic antibodies Casirivimab and Imdevimab. B cell and T cell responses were significantly induced in controls but not in antibody-treated patients. The data indicates that humoral immunity, i. e. high levels of antibodies, negatively impacts reactive immunity, resulting in blunted cellular responses upon boosting. This argues for temporal separation of vaccination efforts; with active vaccination preferably applied before prophylactic therapeutic antibody treatment.

IL-6 Signaling Attenuates TNF-α Production by Plasmacytoid Dendritic Cells in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is characterized by autoimmune joint destruction with debilitating consequences. Despite treatment advancements with biologic therapies, a significant proportion of RA patients show an inadequate clinical response, and restoration of immune self-tolerance represents an unmet therapeutic need. We have previously described a tolerogenic phenotype of plasmacytoid dendritic cells (pDCs) in RA patients responding to anti-TNF-α agents. However, the molecular mechanisms involved in tolerogenic reprogramming of pDCs in RA remain elusive. In this study, guided by transcriptomic analysis of CD303+CD123+ pDCs from RA patients in remission, we revealed enhanced expression of IL-6R and its downstream signaling compared with healthy pDCs. Functional assessment demonstrated that IL-6R engagement resulted in marked reduction of TNF-α secretion by pDCs whereas intracellular TNF-α was significantly increased. Accordingly, pharmacologic inhibition of IL-6R signaling restored TNF-α secretion levels by pDCs. Mechanistic analysis demonstrated impaired activity and decreased lysosomal degradation of ADAM17 (a disintegrin and metalloproteinase 17) sheddase in pDCs, which is essential for TNF-α cleavage. Importantly, reduction of TNF-α secretion by IL-6-treated pDCs attenuated the inflammatory potential of RA patient-derived synovial fibroblasts. Collectively, these findings position pDCs as an important source of TNF-α in RA pathogenesis and unravel an anti-inflammatory mechanism of IL-6 by limiting the pDC-derived TNF-α secretion.

Peripheral blood mononuclear cells are hypomethylated in active rheumatoid arthritis and methylation correlates with disease activity.

OBJECTIVE: Epigenetic modifications are dynamic and influence cellular disease activity. The aim of this study was to investigate global DNA methylation in peripheral blood mononuclear cells (PBMCs) of RA patients to clarify whether global DNA methylation pattern testing might be useful in monitoring disease activity as well as the response to therapeutics. METHODS: Flow cytometric measurement of 5-methyl-cytosine (5'-mC) was established using the cell line U937. In the subsequent prospective study, 62 blood samples were investigated, including 17 healthy donors and 45 RA patients at baseline and after 3 months of treatment with methotrexate, the IL-6 receptor inhibitor sarilumab, and Janus kinase inhibitors. Methylation status was assessed with an anti-5'-mC antibody and analysed in PBMCs and CD4+, CD8+, CD14+ and CD19+ subsets. Signal intensities of 5'-mC were correlated with 28-joint DASs with ESR and CRP (DAS28-ESR and DAS28-CRP). RESULTS: Compared with healthy individuals, PBMCs of RA patients showed a significant global DNA hypomethylation. Signal intensities of 5'-mC correlated with transcription levels of DNMT1, DNMT3B and MTR genes involved in methylation processes. Using flow cytometry, significant good correlations and linear regression values were achieved in RA patients between global methylation levels and DAS28-ESR values for PBMCs (r = -0.55, P = 0.002), lymphocytes (r = -0.57, P = 0.001), CD4+ (r = -0.57, P = 0.001), CD8+ (r = -0.54, P = 0.001), CD14+ (r = -0.49, P = 0.008) and CD19+ (r = -0.52, P = 0.004) cells. CONCLUSIONS: The degree of global DNA methylation was found to be associated with disease activity. Based on this novel approach, the degree of global methylation is a promising biomarker for therapy monitoring and the prediction of therapy outcome in inflammatory diseases.

Dysregulated CD38 Expression on Peripheral Blood Immune Cell Subsets in SLE.

Given its uniformly high expression on plasma cells, CD38 has been considered as a therapeutic target in patients with systemic lupus erythematosus (SLE). Herein, we investigate the distribution of CD38 expression by peripheral blood leukocyte lineages to evaluate the potential therapeutic effect of CD38-targeting antibodies on these immune cell subsets and to delineate the use of CD38 as a biomarker in SLE. We analyzed the expression of CD38 on peripheral blood leukocyte subsets by flow and mass cytometry in two different cohorts, comprising a total of 56 SLE patients. The CD38 expression levels were subsequently correlated across immune cell lineages and subsets, and with clinical and serologic disease parameters of SLE. Compared to healthy controls (HC), CD38 expression levels in SLE were significantly increased on circulating plasmacytoid dendritic cells, CD14++CD16+ monocytes, CD56+ CD16dim natural killer cells, marginal zone-like IgD+CD27+ B cells, and on CD4+ and CD8+ memory T cells. Correlation analyses revealed coordinated CD38 expression between individual innate and memory T cell subsets in SLE but not HC. However, CD38 expression levels were heterogeneous across patients, and no correlation was found between CD38 expression on immune cell subsets and the disease activity index SLEDAI-2K or established serologic and immunological markers of disease activity. In conclusion, we identified widespread changes in CD38 expression on SLE immune cells that highly correlated over different leukocyte subsets within individual patients, but was heterogenous within the population of SLE patients, regardless of disease severity or clinical manifestations. As anti-CD38 treatment is being investigated in SLE, our results may have important implications for the personalized targeting of pathogenic leukocytes by anti-CD38 monoclonal antibodies.

Identification of cross-reactive antibodies for the detection of lymphocytes, myeloid cells and haematopoietic precursors in the naked mole rat.

The naked mole rat (Heterocephalus glaber, NMR) is a rodent with exceptional longevity, low rates of age-related diseases and spontaneous carcinogenesis. The NMR represents an attractive animal model in longevity and cancer research, but there are no NMR-specific antibodies available to study its immune system with respect to age- and cancer-related questions. Substantial homology of major NMR immune cell markers with those of Guinea pig, human and, to a lesser extent, mouse and rat origin are implicated for the existence of immunological cross-reactivity. We identified 10 antibodies recognising eight immunophenotypic markers expressed on the NMR's T and B lymphocytes, macrophages/monocytes and putative haematopoietic precursors and used them for an immunophenotyping of leukocyte subsets of peripheral blood, spleen and bone marrow samples. Overall, we found that the leukocyte composition of NMR peripheral blood is comparable to that of mice. Notably, the frequency of cytotoxic T cells was found to be lower in the NMR compared to corresponding mouse tissues and human blood. Antibodies used in the present paper are available either commercially or from the scientific community and will provide new opportunities for the NMR as a model system in ageing- and cancer-related research areas.

IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases.

B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM(+)CD138(hi)TACI(+)CXCR4(+)CD1d(int)Tim1(int) plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138(+) plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

Mass Cytometry Enabling Absolute and Fast Quantification of Silver Nanoparticle Uptake at the Single Cell Level.

In the last decades, significant efforts have been made to investigate possible cytotoxic effects of metallic nanoparticles (NPs). Methodologies enabling precise information regarding uptake and intracellular distribution of NPs at the single cell level remain to be established. Mass cytometry (MC) has been developed for high-dimensional single cell analyses and is a promising tool to quantify NP-cell interactions. Here, we aim to establish a new MC-based quantification procedure to receive absolute numbers of NPs per single cell by using a calibration that considers the specific transmission efficiency (TE) of suspended NPs. The current MC-quantification strategy accept TE values of complementary metal solutions. In this study, we demonstrate the different transmission behavior of 50 nm silver NPs (AgNP) and silver nitrate solution. We have used identical AgNPs for calibration as for in vitro-differentiated macrophages (THP-1 cell line) in a time- and dose-dependent manner. Our quantification relies on silver intensities measuring AgNPs in the same detection mode as the cells. Results were comparable with the TE quantification strategy using AgNPs but differed when using ionic silver. Furthermore, intact and digested cell aliquots were measured to investigate the impact of MC sample processing on the amount of AgNPs/cell. Taken together, we have provided a MC-specific calibration procedure to precisely calculate absolute numbers of NPs per single cell. Combined with its unique feature of multiplexing up to 50 parameters, MC provides much more information on the single cell level than single cell-inductively coupled plasma mass spectrometry (SC-ICPMS) and, therefore, offers new opportunities in nanotoxicology.

Stabilizing Antibody Cocktails for Mass Cytometry.

Mass cytometry is increasingly employed in larger immune profiling studies involving data acquisitions across several days and multiple sites. For gaining a maximum of information from respective data by computational analyses, several techniques have been developed to minimize noise in mass cytometric data sets, such as sample banking, standardized instrument setup, sample barcoding, and signal normalization. However, the repeated preparation of cocktails composed of isotope-tagged antibodies remained a significant source of error. We here show that premixed antibody cocktails fail to deliver expected staining patterns when stored at 4°C for 4 weeks. As a solution, we developed and tested a cryopreservation method for highly multiplexed antibody cocktails for mass cytometry including lanthanide, palladium, and platinum conjugates that yielded stable staining patterns for at least 9 months when stored at temperatures below -80°C. Using frozen aliquots of antibody cocktails is an economic and flexible approach to significantly improve data consistency in large mass cytometry studies with repetitive staining/measurement cycles spanning several days or involving multiple data acquisition sites. © 2019 International Society for Advancement of Cytometry.

Dual-labelled antibodies for flow and mass cytometry: A new tool for cross-platform comparison and enrichment of target cells for mass cytometry.

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR® labelling protocol. After labelling with 141 Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4+ T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue-142 Nd, CD20-VioGreen-147 Sm, CD27-Cy5-167 Er and CD38-Alexa488-143 Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.

Monocyte alterations in rheumatoid arthritis are dominated by preterm release from bone marrow and prominent triggering in the joint.

OBJECTIVE: Rheumatoid arthritis (RA) accompanies infiltration and activation of monocytes in inflamed joints. We investigated dominant alterations of RA monocytes in bone marrow (BM), blood and inflamed joints. METHODS: CD14+ cells from BM and peripheral blood (PB) of patients with RA and osteoarthritis (OA) were profiled with GeneChip microarrays. Detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and mobilisation. Cytometric profiling determined monocyte subsets of CD14++CD16-, CD14++CD16+ and CD14+CD16+ cells in BM, PB and synovial fluid (SF) and ELISAs quantified the release of activation markers into SF and serum. RESULTS: Investigation of genes differentially expressed between RA and OA monocytes with reference transcriptomes revealed gene patterns of early myeloid precursors in RA-BM and late myeloid precursors along with reduced terminal differentiation to CD14+CD16+monocytes in RA-PB. Patterns associated with tumor necrosis factor/lipopolysaccharide (TNF/LPS) stimulation were weak and more pronounced in RA-PB than RA-BM. Cytometric phenotyping of cells in BM, blood and SF disclosed differences related to monocyte subsets and confirmed the reduced frequency of terminally differentiated CD14+CD16+monocytes in RA-PB. Monocyte activation in SF was characterised by the predominance of CD14++CD16++CD163+HLA-DR+ cells and elevated concentrations of sCD14, sCD163 and S100P. CONCLUSION: Patterns of less mature and less differentiated RA-BM and RA-PB monocytes suggest increased turnover with accelerated monocytopoiesis, BM egress and migration into inflamed joints. Predominant activation in the joint indicates the action of local and primary stimuli, which may also promote adaptive immune triggering through monocytes, potentially leading to new diagnostic and therapeutic strategies.

miR-125b controls monocyte adaptation to inflammation through mitochondrial metabolism and dynamics.

Metabolic changes drive monocyte differentiation and fate. Although abnormal mitochondria metabolism and innate immune responses participate in the pathogenesis of many inflammatory disorders, molecular events regulating mitochondrial activity to control life and death in monocytes remain poorly understood. We show here that, in human monocytes, microRNA-125b (miR-125b) attenuates the mitochondrial respiration through the silencing of the BH3-only proapoptotic protein BIK and promotes the elongation of the mitochondrial network through the targeting of the mitochondrial fission process 1 protein MTP18, leading to apoptosis. Proinflammatory activation of monocyte-derived macrophages is associated with a concomitant increase in miR-125b expression and decrease in BIK and MTP18 expression, which lead to reduced oxidative phosphorylation and enhanced mitochondrial fusion. In a chronic inflammatory systemic disorder, CD14+ blood monocytes display reduced miR-125b expression as compared with healthy controls, inversely correlated with BIK and MTP18 messenger RNA expression. Our findings not only identify BIK and MTP18 as novel targets for miR-125b that control mitochondrial metabolism and dynamics, respectively, but also reveal a novel function for miR-125b in regulating metabolic adaptation of monocytes to inflammation. Together, these data unravel new molecular mechanisms for a proapoptotic role of miR-125b in monocytes and identify potential targets for interfering with excessive inflammatory activation of monocytes in inflammatory disorders.

Silver nanoparticles for the detection of cell surface antigens in mass cytometry.

Mass cytometry has pioneered >40-parameter single-cell analyses that allow for the characterization of complex cellular networks at unprecedented depth. Up to 135 parameters can be simultaneously detected, but limited availability of metal tags suitable for labeling of specific probes prevents optimal exploitation of the analytical capacity of mass cytometers. To this end, we here establish the application of elemental silver nanoparticles (AgNP) of different size for reporting cell surface antigens on human leukocytes in mass cytometry assays. The mass channels at 107 Da and 109 Da are uniquely occupied by silver isotopes and do not interfere with other mass cytometry reagents. Streptavidin-coated AgNP (SA-AgNP) facilitated distinct and specific detection of various antigens, such as CD8, CD244 and CD294 on peripheral blood leukocytes pre-incubated with respective biotinylated primary antibodies. Signal intensities elicited by 40 nm-sized AgNP allowed specific detection of the low abundance antigen CD25 on both, peripheral blood regulatory T cells and CD25lo CD127+ CD4+ T cells, enabling their distinct clustering in viSNE plots. SA-AgNP were of high elemental purity, showed minor background binding to cells in immunoassays, and were compatible with previously established staining protocols for PBMC and leukocytes, facilitating their use in complex mass cytometry panels. Considering the synthesis of AgNP from isotopically purified silver, the usage of AgNP extends the analytical capacity of mass cytometry panels by one, prospectively two, additional parameters, suitable for the detection of cellular targets of low abundance. © 2016 International Society for Advancement of Cytometry.

Are interferon-related biomarkers advantageous for monitoring disease activity in systemic lupus erythematosus? A longitudinal benchmark study.

Objective: To determine the clinical value of six traditional and three IFN-related biomarkers in monitoring disease activity (DA) in SLE. Methods: Prospective longitudinal study of IFNα, IFNγ-inducible protein 10 (IP-10) and sialic acid-binding Ig-like lectin 1 (SIGLEC1) vs antibodies against dsDNA (ELISA and Farr radioimmunoassay), dsDNA-complexed nucleosomes (anti-dsDNA-NcX: ELISA), nucleosomes (ANuA: ELISA) and complement C3/C4 for correlation with DA (measured by BILAG 2004 index) in 26 SLE patients (77 visits). Optimal upper and lower longitudinal thresholds for the biomarkers and their accuracies for reflecting clinically relevant changes in DA (flares and remission) were determined by receiver operating characteristic and Youden index analysis. Results: Increases in IP-10, SIGLEC1 and ANuA to + 101.6 pg/ml, +5.01 relative mean fluorescence intensity and +16.20 IU/ml above the calculated upper longitudinal threshold significantly reflected lupus flares, with a sensitivity and specificity of 50 and 95% for IP-10, 83 and 90% for SIGLEC1 and 58 and 95% for ANuA. Decreases in anti-dsDNA (ELISA), IFNα and anti-dsDNA (Farr assay) to - 64.7 IU/ml, -16.69 pg/ml and -3.3 IU/ml below lower longitudinal thresholds, respectively, best reflected remission, with sensitivity and specificity of 75 and 95%, 62 and 90%, and 75 and 90%, respectively. Conclusion: IP-10, SIGLEC1 and ANuA emerged as advantageous biomarkers for monitoring disease activity. This is the first study in SLE that provides longitudinal biomarker thresholds and test accuracies for SLE flares and remitting disease. In the context of IFN-directed therapies, chemokines and fluorescence-activated cell sorting-based IFN biomarkers for monitoring SLE activity should be further studied.

De novo-induced self-antigen-specific Foxp3+ regulatory T cells impair the accumulation of inflammatory dendritic cells in draining lymph nodes.

Foxp3(+) regulatory T cell (Treg)-based immunotherapy holds promise for autoimmune diseases. However, this effort has been hampered by major caveats, including the low frequency of autoantigen-specific Foxp3(+) Tregs and lack of understanding of their molecular and cellular targets, in an unmanipulated wild-type (WT) immune repertoire. In this study, we demonstrate that infusion of myelin in WT mice results in the de novo induction of myelin-specific Foxp3(+) Tregs in WT mice and amelioration of experimental autoimmune encephalomyelitis. Myelin-specific Foxp3(+) Tregs exerted their effect both by diminishing Ag-bearing inflammatory dendritic cell (iDC) recruitment to lymph nodes and by impairing their function. Transcriptome analysis of ex vivo-isolated Treg-exposed iDCs showed significant enrichment of transcripts involved in functional properties of iDCs, including chemotaxis-related genes. To this end, CCR7 expression by iDCs was significantly downregulated in tolerant mice and this was tightly regulated by the presence of IL-10. Collectively, our data demonstrate a novel model for deciphering the Ag-specific Foxp3(+) Treg-mediated mechanisms of tolerance and delineate iDCs as a Foxp3(+) Treg cellular target in unmanipulated mice.

Low Thymic Activity and Dendritic Cell Numbers Are Associated with the Immune Response to Primary Viral Infection in Elderly Humans.

Immunological competence declines progressively with age, resulting in increased susceptibility of the elderly to infection and impaired responses to vaccines. Underlying mechanisms remain largely obscure as they have been related to complex, individual systemic immune properties that are challenging to investigate. In this study, we explored age-related changes in human immunity during a primary virus infection experimentally induced by immunization with live-attenuated yellow fever (YF) vaccine. Applying detailed serology, advanced FACS analysis, and systems biology, we discovered that aged subjects developed fewer neutralizing Abs, mounted diminished YF-specific CD8(+) T cell responses, and showed quantitatively and qualitatively altered YF-specific CD4(+) T cell immunity. Among numerous immune signatures, low in vivo numbers of naive CD4(+) recent thymic emigrants and peripheral dendritic cells correlated well with reduced acute responsiveness and altered long-term persistence of human cellular immunity to YF vaccination. Hence, we reveal in this article that essential elements of immune responses such as recent thymic emigrants and dendritic cells strongly relate to productive immunity in the elderly, providing a conceivable explanation for diminished responsiveness to vaccination with neoantigens and infection with de novo pathogens in the aged population.

Rsk2 controls synovial fibroblast hyperplasia and the course of arthritis.

OBJECTIVE: Arthritis is a chronic inflammatory disease characterised by immune cell infiltration and mesenchymal cell expansion in the joints. Although the role of immune cells in arthritis is well characterised, the development of mesenchymal cell hyperplasia needs to be better defined. Here, we analysed the role of the ribosomal S6 kinase Rsk2, which we found to be highly activated in joints of patients with arthritis, in the development of mesenchymal cell hyperplasia. METHODS: We genetically inactivated Rsk2 in the tumour necrosis factor (TNF)-α transgenic (TNFtg) mice, an animal model for human inflammatory arthritis. Clinical and histological signs of arthritis as well as molecular markers of inflammation and joint destruction were quantified. Fibroblast-like synoviocytes (FLS) were characterised in vitro and the effect of Rsk2 deletion on the pattern of gene expression was determined. RESULTS: Rsk2 deficiency in TNFtg mice results in earlier and exacerbated inflammation as well as increased bone and cartilage destruction. The production of inflammatory cytokines, matrix metalloproteinases and osteoclastogenic molecules was significantly increased in vivo upon Rsk2 inactivation. Bone marrow deficient in Rsk2 could not transfer this phenotype, indicating that Rsk2 expression in mesenchymal cells controls the course of arthritis. Indeed, Rsk2 deficiency was associated with a more activated phenotype and higher proliferative capacity of FLS, thereby increasing cytokines and production of matrix proteinases. CONCLUSIONS: Rsk2 emerges as a key regulator of mesenchymal cell numbers in the joint and thereby could be targeted to control the inflammatory and tissue-destructive feature of joints in arthritis.

immunoClust--An automated analysis pipeline for the identification of immunophenotypic signatures in high-dimensional cytometric datasets.

Multiparametric fluorescence and mass cytometry offers new perspectives to disclose and to monitor the high diversity of cell populations in the peripheral blood for biomarker research. While high-end cytometric devices are currently available to detect theoretically up to 120 individual parameters at the single cell level, software tools are needed to analyze these complex datasets automatically in acceptable time and without operator bias or knowledge. We developed an automated analysis pipeline, immunoClust, for uncompensated fluorescence and mass cytometry data, which consists of two parts. First, cell events of each sample are grouped into individual clusters. Subsequently, a classification algorithm assorts these cell event clusters into populations comparable between different samples. The clustering of cell events is designed for datasets with large event counts in high dimensions as a global unsupervised method, sensitive to identify rare cell types even when next to large populations. Both parts use model-based clustering with an iterative expectation maximization algorithm and the integrated classification likelihood to obtain the clusters. A detailed description of both algorithms is presented. Testing and validation was performed using 1) blood cell samples of defined composition that were depleted of particular cell subsets by magnetic cell sorting, 2) datasets of the FlowCAP III challenges to identify populations of rare cell types and 3) high-dimensional fluorescence and mass-cytometry datasets for comparison with conventional manual gating procedures. In conclusion, the immunoClust-algorithm is a promising tool to standardize and automate the analysis of high-dimensional cytometric datasets. As a prerequisite for interpretation of such data, it will support our efforts in developing immunological biomarkers for chronic inflammatory disorders and therapy recommendations in personalized medicine. immunoClust is implemented as an R-package and is provided as source code from www.bioconductor.org.

IFNα and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus.

OBJECTIVES: To evaluate and compare the clinical efficacy of three biomarkers for interferon (IFN) activity (measured directly and indirectly) and six traditional biomarkers in indicating current and prospective disease activity (DA) in systemic lupus erythematosus (SLE). METHODS: IFNα (dissociation-enhanced lanthanide fluorescent immunoassay), IFNγ-inducible protein 10 (IP-10) (ELISA) and sialic acid-binding Ig-like lectin 1 (SIGLEC-1) (flow cytometry) were measured in 79 accurately characterised patients with lupus and compared with serum titres of Anti-dsDNA (ELISA and radioimmunoassay), Anti-dsDNA-NcX ELISA, Anti-Nuc ELISA, and complement C3 and C4. DA was evaluated using the British Isles Lupus Assessment Group 2004 Index (BILAG-2004) and a modified SLE Disease Activity Index-2000 (mSLEDAI-2K). In addition, 31 clinically quiescent patients were monitored for flares over the course of 180 days. RESULTS: Increased levels of IFNα, IP-10 and SIGLEC-1 were found in 32%, 50% and 86%, respectively, of 66 patients with active SLE. IFNα (r=0.45; p<0.0001) and SIGLEC-1 (r=0.54; p<0.0001) correlated better with BILAG-2004 than did IP-10 (r=0.38; p=0.0002), Farr assay (r=0.40; p=0.0001), Anti-dsDNA-NcX ELISA (r=0.28; p=0.0061), Anti-dsDNA ELISA (r=0.31; p=0.0025), Anti-Nuc ELISA (r=0.25; p=0.0121), C3 (r=-0.43; p<0.0001) and C4 (r=-0.33; p=0.0013). Predictors of SLE flares were disease duration ≤92 months, mild clinical activity (in contrast with no activity), complement C3≤89 mg/dl and IFNα≥20 pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. CONCLUSIONS: IFNα, IP-10 and SIGLEC-1 emerged as beneficial biomarkers of DA in patients with SLE. Therefore the implementation of IFN biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-IFN-directed therapies.