The fate of immune complexes in membranous nephropathy.

The most characteristic feature of membranous nephropathy (MN) is the presence of subepithelial electron dense deposits and the consequential thickening of the glomerular basement membrane. There have been great advances in the understanding of the destiny of immune complexes in MN by the benefit of experimental models represented by Heymann nephritis. Subepithelial immune complexes are formed in situ by autoantibodies targeting native autoantigens or exogenous planted antigens such as the phospholipase A2 receptor (PLA2R) and cationic BSA respectively. The nascent immune complexes would not be pathogenic until they develop into immune deposits. Podocytes are the major source of autoantigens in idiopathic membranous nephropathy. They also participate in the modulation and removal of the immune complexes to a large extent. The balance between deposition and clearance is regulated by a wide range of factors such as the composition and physicochemical properties of the immune complexes and the complement system. Complement components such as C3 and C1q have been reported to be precipitated with the deposits whereas a complement regulatory protein CR1 expressed by podocytes is involved in the phagocytosis of immune complexes by podocytes. Podocytes regulate the dynamic change of immune complexes which is disturbed in membranous nephropathy. To elucidate the precise fate of the immune complexes is essential for developing more rational and novel therapies for membranous nephropathy.

Fcγ-receptor-IIIA bioactivity of circulating and synovial immune complexes in rheumatoid arthritis.

OBJECTIVE: Previous technical limitations prevented the proof of Fcγ-receptor (FcγR)-activation by soluble immune complexes (sICs) in patients. FcγRIIIa (CD16) is a risk factor in rheumatoid arthritis (RA). We aimed at determining the presence of CD16-activating sICs in RA and control diseases. METHODS: Sera from an exploratory cohort (n=50 patients with RA) and a validation cohort (n=106 patients with RA, 20 patients with psoriasis arthritis (PsA), 22 patients with systemic lupus erythematosus (SLE) and 31 healthy controls) were analysed using a new reporter cell assay. Additionally, 26 synovial fluid samples were analysed, including paired serum/synovial samples. RESULTS: For the first time using a reliable and sensitive functional assay, the presence of sICs in RA sera was confirmed. sICs possess an intrinsic capacity to activate CD16 and can be found in both synovial fluid and in blood. In low experimental dilutions, circulating sICs were also detected in a subset of healthy people and in PsA. However, we report a significantly increased frequency of bioactive circulating sICs in RA. While the bioactivity of circulating sICs was low and did not correlate with clinical parameters, synovial sICs were highly bioactive and correlated with serum autoantibody levels. Receiver operator curves indicated that sICs bioactivity in synovial fluid could be used to discriminate immune complex-associated arthritis from non-associated forms. Finally, circulating sICs were more frequently found in SLE than in RA. The degree of CD16 bioactivity showed strong donor-dependent differences, especially in SLE. CONCLUSIONS: RA is characterised by the presence of circulating and synovial sICs that can engage and activate CD16.

Lupus IgA1 autoantibodies synergize with IgG to enhance plasmacytoid dendritic cell responses to RNA-containing immune complexes.

Autoantibodies to nuclear antigens are hallmarks of systemic lupus erythematosus (SLE) where they contribute to pathogenesis. However, there remains a gap in our knowledge regarding how different isotypes of autoantibodies contribute to this autoimmune disease, including the production of the critical type I interferon (IFN) cytokines by plasmacytoid dendritic cells (pDCs) in response to immune complexes (ICs). We focused on IgA, which is the second-most prevalent isotype in serum and, along with IgG, is deposited in glomeruli in individuals with lupus nephritis. We show that individuals with SLE have serum IgA autoantibodies against most nuclear antigens, correlating with IgG against the same antigen. We investigated whether IgA autoantibodies against a major SLE autoantigen, Smith ribonucleoprotein (Sm/RNP), played a role in IC activation of pDCs. We found that pDCs expressed the IgA-specific Fc receptor, FcαR, and IgA1 autoantibodies synergized with IgG in RNA-containing ICs to generate robust primary blood pDC IFN-α responses in vitro. pDC responses to these ICs required both FcαR and FcγRIIa, showing synergy between these Fc receptors. Sm/RNP IC binding to and internalization by pDCs were greater when ICs contained both IgA1 and IgG. Circulating pDCs from individuals with SLE had higher binding of IgA1-containing ICs and higher expression of FcαR than pDCs from healthy control individuals. Although pDC FcαR expression correlated with the blood IFN-stimulated gene signature in SLE, Toll-like receptor 7 agonists, but not IFN-α, up-regulated pDC FcαR expression in vitro. Together, we show a mechanism by which IgA1 autoantibodies contribute to SLE pathogenesis.

Exploiting the Role of Features for Antigens-Antibodies Interaction Site Prediction.

Antibodies are a class of proteins that recognize and neutralize pathogens by binding to their antigens. They are the most significant category of biopharmaceuticals for both diagnostic and therapeutic applications. Understanding how antibodies interact with their antigens plays a fundamental role in drug and vaccine design and helps to comprise the complex antigen binding mechanisms. Computational methods for predicting interaction sites of antibody-antigen are of great value due to the overall cost of experimental methods. Machine learning methods and deep learning techniques obtained promising results.In this work, we predict antibody interaction interface sites by applying HSS-PPI, a hybrid method defined to predict the interface sites of general proteins. The approach abstracts the proteins in terms of hierarchical representation and uses a graph convolutional network to classify the amino acids between interface and non-interface. Moreover, we also equipped the amino acids with different sets of physicochemical features together with structural ones to describe the residues. Analyzing the results, we observe that the structural features play a fundamental role in the amino acid descriptions. We compare the obtained performances, evaluated using standard metrics, with the ones obtained with SVM with 3D Zernike descriptors, Parapred, Paratome, and Antibody i-Patch.

Machine-learning-based structural analysis of interactions between antibodies and antigens.

Computational analysis of paratope-epitope interactions between antibodies and their corresponding antigens can facilitate our understanding of the molecular mechanism underlying humoral immunity and boost the design of new therapeutics for many diseases. The recent breakthrough in artificial intelligence has made it possible to predict protein-protein interactions and model their structures. Unfortunately, detecting antigen-binding sites associated with a specific antibody is still a challenging problem. To tackle this challenge, we implemented a deep learning model to characterize interaction patterns between antibodies and their corresponding antigens. With high accuracy, our model can distinguish between antibody-antigen complexes and other types of protein-protein complexes. More intriguingly, we can identify antigens from other common protein binding regions with an accuracy of higher than 70% even if we only have the epitope information. This indicates that antigens have distinct features on their surface that antibodies can recognize. Additionally, our model was unable to predict the partnerships between antibodies and their particular antigens. This result suggests that one antigen may be targeted by more than one antibody and that antibodies may bind to previously unidentified proteins. Taken together, our results support the precision of antibody-antigen interactions while also suggesting positive future progress in the prediction of specific pairing.

Hexamerization: explaining the original sin of IgG-mediated complement activation in acute lung injury.

Although antibody-mediated lung damage is a major factor in transfusion-related acute lung injury (ALI), autoimmune lung disease (for example, coatomer subunit α [COPA] syndrome), and primary graft dysfunction following lung transplantation, the mechanism by which antigen-antibody complexes activate complement to induce lung damage remains unclear. In this issue of the JCI, Cleary and colleagues utilized several approaches to demonstrate that IgG forms hexamers with MHC class I alloantibodies. This hexamerization served as a key pathophysiological mechanism in alloimmune lung injury models and was mediated through the classical pathway of complement activation. Additionally, the authors provided avenues for exploring therapeutics for this currently hard-to-treat clinical entity that has several etiologies but a potentially focused mechanism.

Inflammatory Profiles Induced by Intranasal Immunization with Ricin Toxin-immune Complexes.

The underlying contribution of immune complexes in modulating adaptive immunity in mucosal tissues remains poorly understood. In this report, we examined, in mice, the proinflammatory response elicited by intranasal delivery of the biothreat agent ricin toxin (RT) in association with two toxin-neutralizing mAbs, SylH3 and PB10. We previously demonstrated that ricin-immune complexes (RICs) induce the rapid onset of high-titer toxin-neutralizing Abs that persist for months. We now demonstrate that such responses are dependent on CD4+ T cell help, because treatment of mice with an anti-CD4 mAb abrogated the onset of RT-specific Abs following intranasal RICs exposure. To define the inflammatory environment associated with RIC exposure, we collected bronchoalveolar lavage fluid (BALF) and sera from mice 6, 12, and 18 h after they had received RT or RICs by the intranasal route. A 32-plex cytometric bead array revealed an inflammatory profile elicited by RT that was dominated by IL-6 (>1500-fold increase in BALF) and secondarily by KC (CXCL1), G-CSF, GM-CSF, and MCP-1. RICs induced inflammatory profiles in both BALF and serum response that were similar to RT, albeit at markedly reduced levels. These results demonstrate that RICs retain the capacity to induce local and systemic inflammatory cytokines/chemokines that, in turn, may influence Ag sampling and presentation in the lung mucosa and draining lymph nodes. A better understanding of the fate of immune complexes following intranasal delivery has implications for the development of mucosal vaccines for biothreats and emerging infectious diseases.

Elevated Complement Activation Fragments and C1q-Binding Circulating Immune Complexes in Varied Phases of Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is a causative agent of a disease continuum, ranging from an acute transient chikungunya fever to chronic incapacitating viral arthralgia. The interaction between anti-CHIKV antibodies and the complement system has recently received attention. However, the contribution of complement activation in CHIKV-induced pathologies has not been fully elucidated. The present study was undertaken to delineate the possible contribution of complement activation in CHIKV-induced disease progression. In this study, using plasma specimens of chikungunya patients in the acute, chronic, and recovered phases of infection, we explicated the involvement of complement activation in CHIKV disease progression by ELISAs and Bio-Plex assays. Correlation analysis was carried out to demonstrate interrelation among C1q-binding IgG-containing circulating immune complexes (CIC-C1q), complement activation fragments (C3a, C5a, sC5b-9), and complement-modulated pro-inflammatory cytokines (IL-1ß, IL-18, IL-6, and TNF-α). We detected elevated complement activation fragments, CIC-C1q, and complement-modulated cytokines in the varied patient groups compared with the healthy controls, indicating persistent activation of the complement system. Furthermore, we observed statistically significant correlations among CIC-C1q with complement activation fragments and C3a with complement modulatory cytokines IL-1ß, IL-6, and IL-18 during the CHIKV disease progression. Taken together, the current data provide insight into the plausible association between CICs, complement activation, subsequent complement modulatory cytokine expression, and CHIKV etiopathology.

Decreased IgA binding levels were accompanied by increased IgG sialylation in IgA nephropathy.

INTRODUCTION: IgA nephropathy (IgAN) is a kidney disorder characterized by the deposition of circulating immune complexes of IgG bound to galactose-deficient IgA1 (Gd-IgA1) in the mesangial glomeruli. However, limited research has been conducted on the levels of IgA binding in relation to the various sialylation profiles of IgG in IgAN. MATERIALS AND METHODS: Sialylated IgG (SA-IgG) and desialylated IgG (DSA-IgG) were isolated from IgAN patients. The IgG-IgA immune complex (IgG-IgA-IC) was detected using two customized commercial ELISA kits. Additionally, IgG was enzymatically digested with neuraminidase to produce DSA-IgG. Subsequently, the binding capacities of both intact IgG and the neuraminidase-digested DSA-IgG with Gd-IgA1 were determined using ELISA kits. RESULTS: Our research revealed that SA-IgG levels were negatively correlated with Gd-IgA1 (R = -0.16, p = 0.03) in IgAN patients. The optical density (OD) levels of IgG-IgA complexes in SA-IgG samples were significantly lower (0.58 ± 0.09) compared to those in DSA-IgG samples (0.78 ± 0.12) when using the Gd-IgA1 assay kit. These results were confirmed using an IgG assay kit, which showed that the SA-IgG groups had significantly lower IgA indices (0.31 ± 0.12) compared to the DSA-IgG groups (0.57 ± 0.19). Furthermore, we investigated the binding capacity of IgG with different sialic acid levels to Gd-IgA1. The results revealed that neuraminidase digestion of IgG increased its propensity to bind to Gd-IgA1. Additionally, we examined the binding capacity of both intact IgG and DSA-IgG to Gd-IgA1 at different mix ratios (IgG 1.5 µg and Gd-IgA1 1.5 µg, IgG 1.5 µg and Gd-IgA1 3 µg, IgG 3 µg and Gd-IgA1 1.5 µg). Interestingly, DSA-IgG demonstrated significantly higher binding capacity to Gd-IgA1 compared to intact IgG at all mix ratios tested. CONCLUSION: The preliminary findings from our present study indicate that the binding level of IgA in purified sialylated IgG is lower than that in desialylated IgG.

Prediction of Paratope-Epitope Pairs Using Convolutional Neural Networks.

Antibodies play a central role in the adaptive immune response of vertebrates through the specific recognition of exogenous or endogenous antigens. The rational design of antibodies has a wide range of biotechnological and medical applications, such as in disease diagnosis and treatment. However, there are currently no reliable methods for predicting the antibodies that recognize a specific antigen region (or epitope) and, conversely, epitopes that recognize the binding region of a given antibody (or paratope). To fill this gap, we developed ImaPEp, a machine learning-based tool for predicting the binding probability of paratope-epitope pairs, where the epitope and paratope patches were simplified into interacting two-dimensional patches, which were colored according to the values of selected features, and pixelated. The specific recognition of an epitope image by a paratope image was achieved by using a convolutional neural network-based model, which was trained on a set of two-dimensional paratope-epitope images derived from experimental structures of antibody-antigen complexes. Our method achieves good performances in terms of cross-validation with a balanced accuracy of 0.8. Finally, we showcase examples of application of ImaPep, including extensive screening of large libraries to identify paratope candidates that bind to a selected epitope, and rescoring and refining antibody-antigen docking poses.

Recombinant ADAMTS13 for Immune Thrombotic Thrombocytopenic Purpura.

In patients with immune thrombotic thrombocytopenic purpura (iTTP), autoantibodies against the metalloprotease ADAMTS13 lead to catastrophic microvascular thrombosis. However, the potential benefits of recombinant human ADAMTS13 (rADAMTS13) in patients with iTTP remain unknown. Here, we report the clinical use of rADAMTS13, which resulted in the rapid suppression of disease activity and complete recovery in a critically ill patient whose condition had proved to be refractory to all available treatments. We also show that rADAMTS13 causes immune complex formation, which saturates the autoantibody and may promote its clearance. Our data support the role of rADAMTS13 as a novel adjunctive therapy in patients with iTTP.

DNA Nanostructure-Templated Antibody Complexes Provide Insights into the Geometric Requirements of Human Complement Cascade Activation.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

Antibody-drug conjugate adverse effects can be understood and addressed based on immune complex clearance mechanisms.

ABSTRACT: Numerous antibody-drug conjugates (ADCs) are being developed for cancer immunotherapy. Although several of these agents have demonstrated considerable clinical efficacy and have won Food and Drug Administration (FDA) approval, in many instances, they have been characterized by adverse side effects (ASEs), which can be quite severe in a fraction of treated patients. The key hypothesis in this perspective is that many of the most serious ASEs associated with the use of ADCs in the treatment of cancer can be most readily explained and understood due to the inappropriate processing of these ADCs via pathways normally followed for immune complex clearance, which include phagocytosis and trogocytosis. We review the key published basic science experiments and clinical observations that support this idea. We propose that it is the interaction of the ADC with Fcγ receptors expressed on off-target cells and tissues that can most readily explain ADC-mediated pathologies, which therefore provides a rationale for the design of protocols to minimize ASEs. We describe measurements that should help identify those patients most likely to experience ASE due to ADC, and we propose readily available treatments as well as therapies under development for other indications that should substantially reduce ASE associated with ADC. Our focus will be on the following FDA-approved ADC for which there are substantial literatures: gemtuzumab ozogamicin and inotuzumab ozogamicin; and trastuzumab emtansine and trastuzumab deruxtecan.

Unique Interplay Between Antinuclear Antibodies and Nuclear Molecules in the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that primarily affects young women and causes a wide range of inflammatory manifestations. The hallmark of SLE is the production of antibodies to components of the cell nucleus (antinuclear antibodies [ANAs]). These antibodies can bind to DNA, RNA, and protein complexes with nucleic acids. Among ANAs, antibodies to DNA (anti-DNA) are markers for classification and disease activity, waxing and waning disease activity in many patients. In the blood, anti-DNA antibodies can bind to DNA to form immune complexes with two distinct roles in pathogenesis: (1) renal deposition to provoke nephritis and (2) stimulation of cytokine production following uptake into innate immune cells and interaction with internal nucleic acid sensors. These sensors are part of an internal host defense system in the cell cytoplasm that can respond to DNA from infecting organisms; during cell stress, DNA from nuclear and mitochondrial sources can also trigger these sensors. The formation of immune complexes requires a source of extracellular DNA in an immunologically accessible form. As shown in in vivo and in vitro systems, extracellular DNA can emerge from dead and dying cells in both a free and a particulate form. Neutrophils undergoing the process of NETosis can release DNA in mesh-like structures called neutrophil extracellular traps. In SLE, therefore, the combination of ANAs and immunologically active DNA can create new structures that can promote inflammation throughout the body as well as drive organ inflammation and damage.

Novel assay format for total anti-adeno-associated virus antibody detection with low capsid consumption and built-in specificity control.

Aim: To develop an assay format for detection of total anti-adeno-associated virus 2 (AAV2) antibodies with low capsid material consumption. Methods: An immune complex (IC) assay format was developed. The format is based on the formation of ICs in solution and their subsequent detection using an anti-AAV2 antibody for capture and an antibody against the study species IgG for detection. Results: The feasibility of the IC assay for detection of preexisting and treatment-emergent anti-AAV2 antibodies was demonstrated in cynomolgus monkey and human serum samples, including samples from a preclinical study with AAV2-based therapies. Conclusion: The presented IC assay is an easy-to-perform total anti-AAV2 antibody assay that requires a small amount of unlabeled capsid material and provides an intrinsic specificity control.

[Box: see text].

Internalization of HIV-1 by Phagocytes Is Increased When Virions Are Opsonized with Multimeric Antibody in the Presence of Complement.

The low abundance of envelope spikes and the inability of IgG to aggregate virions render HIV-1 an inadequate target for antibody-mediated clearance by phagocytes. In an attempt to improve the ability of antibody to mediate the internalization of HIV-1 virions, we generated multimers of the broadly neutralizing HIV-1-specific monoclonal antibody (MAb) VRC01 using site-directed mutagenesis of the Fc segment. We then measured virion internalization using primary human monocytes and neutrophils. We found that, in the absence of complement, immune complexes consisting of HIV-1 virions and VRC01 multimers were slightly more efficiently internalized than were complexes formed with monomeric VRC01. The presence of complement, however, greatly augmented internalization of immune complexes formed with the multimeric MAb but had little impact on monomeric MAb-mediated internalization. Multimerization and the presence of complement overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions and may thus provide a therapeutic approach to clearing virus. IMPORTANCE Antibody-mediated internalization of HIV-1 by phagocytes, a potential mechanism for clearing virus, is very inefficient. In an effort to improve viral clearance, we produced a multimeric form of the broadly neutralizing monoclonal antibody VRC01. We found that VRC01 antibody multimers (primarily hexamers) were only slightly more efficient in mediating HIV-1 internalization than was monomeric VRC01. However, the addition of complement resulted in substantially greater internalization of multimer-opsonized virus. In contrast, complement had little if any impact on internalization of monomer-opsonized virus. Therefore, antibody multimerization in combination with complement may overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions. Our findings may provide a therapeutic approach to clearing virus.

Impact of anti-PEG antibody affinity on accelerated blood clearance of pegylated epoetin beta in mice.

Antibodies that bind polyethylene glycol (PEG) can be induced by pegylated biomolecules and also exist in a significant fraction of healthy individuals who have never received pegylated medicines. The binding affinity of antibodies against PEG (anti-PEG antibodies) likely varies depending on if they are induced or naturally occurring. Anti-PEG antibodies can accelerate the clearance of pegylated medicines from the circulation, resulting in loss of drug efficacy, but it is unknown how accelerated blood clearance is affected by anti-PEG antibody affinity. We identified a panel of anti-PEG IgG and IgM antibodies with binding avidities ranging over several orders of magnitude to methoxy polyethylene glycol-epoetin beta (PEG-EPO), which is used to treat patients suffering from anemia. Formation of in vitro immune complexes between PEG-EPO and anti-PEG IgG or IgM antibodies was more obvious as antibody affinity increased. Likewise, high affinity anti-PEG antibodies produced greater accelerated blood clearance of PEG-EPO as compared to low affinity antibodies. The molar ratio of anti-PEG antibody to PEG-EPO that accelerates drug clearance in mice correlates with antibody binding avidity. Our study indicates that the bioactivity of PEG-EPO may be reduced due to rapid clearance in patients with either high concentrations of low affinity or low concentrations of high affinity anti-PEG IgG and IgM antibodies.

Immune Complex Formation Is Associated With Loss of Tolerance and an Antibody Response to Both Drug and Target.

AMG 966 is a bi-specific, heteroimmunoglobulin molecule that binds both tumor necrosis factor alpha (TNFα) and TNF-like ligand 1A (TL1A). In a first-in-human clinical study in healthy volunteers, AMG 966 elicited anti-drug antibodies (ADA) in 53 of 54 subjects (98.1%), despite a paucity of T cell epitopes observed in T cell assays. ADA were neutralizing and bound to all domains of AMG 966. Development of ADA correlated with loss of exposure. In vitro studies demonstrated that at certain drug-to-target ratios, AMG 966 forms large immune complexes with TNFα and TL1A, partially restoring the ability of the aglycosylated Fc domain to bind FcγRIa and FcγRIIa, leading to the formation of ADA. In addition to ADA against AMG 966, antibodies to endogenous TNFα were also detected in the sera of subjects dosed with AMG 966. This suggests that the formation of immune complexes between a therapeutic and target can cause loss of tolerance and elicit an antibody response against the target.

RNA-Containing Immune Complexes Formed by Anti-Melanoma Differentiation Associated Gene 5 Autoantibody Are Potent Inducers of IFN-α.

Objective: Anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is a distinctive serology hallmark of dermatomyositis (DM). As an autoantigen, MDA5 is a cytoplasmic RNA recognition receptor. The aim of this study was to address the question of whether the RNA-containing immune complex (IC) formed by MDA5 and anti-MDA5 could activate type I interferon (IFN) response. Method: Patients with anti-MDA5+ DM (n = 217), anti-MDA5- DM (n = 68), anti-synthase syndrome (ASyS, n = 57), systemic lupus erythematosus (SLE, n = 245), rheumatoid arthritis (RA, n = 89), and systemic sclerosis (SSc, n = 30) and healthy donors (HD, n = 94) were enrolled in our studies. Anti-MDA5 antibody was detected by line blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. Cytokine profiling was determined by multiplex flow cytometry, and IFN-α was further measured by ELISA. Type I IFN-inducible genes were detected by quantitative PCR (qPCR). RNA-IC binding was analyzed by RNA immunoprecipitation. Plasmacytoid dendritic cells (pDCs) derived from healthy donors were cultivated and stimulated with MDA5 ICs with or without RNase and Toll-like receptor 7 (TLR-7) agonist. The interaction between MDA5 ICs and TLR7 was evaluated by immunoprecipitation and confocal microscopy. Results: According to our in-house ELISA, the presence of anti-MDA5 antibody in 76.1% of DM patients, along with 14.3% of SLE patients who had a lower titer yet positive anti-MDA5 antibody, was related to the high level of peripheral IFN-α. ICs formed by MDA5 and anti-MDA5 were potent inducers of IFN-α via TLR-7 in an RNA-dependent manner in vitro. Conclusion: Our data provided evidence of the mechanistic relevance between the anti-MDA5 antibody and type I IFN pathway.