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.

Unsupervised evolution of protein and antibody complexes with a structure-informed language model.

Large language models trained on sequence information alone can learn high-level principles of protein design. However, beyond sequence, the three-dimensional structures of proteins determine their specific function, activity, and evolvability. Here, we show that a general protein language model augmented with protein structure backbone coordinates can guide evolution for diverse proteins without the need to model individual functional tasks. We also demonstrate that ESM-IF1, which was only trained on single-chain structures, can be extended to engineer protein complexes. Using this approach, we screened about 30 variants of two therapeutic clinical antibodies used to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We achieved up to 25-fold improvement in neutralization and 37-fold improvement in affinity against antibody-escaped viral variants of concern BQ.1.1 and XBB.1.5, respectively. These findings highlight the advantage of integrating structural information to identify efficient protein evolution trajectories without requiring any task-specific training data.

AttABseq: an attention-based deep learning prediction method for antigen-antibody binding affinity changes based on protein sequences.

The optimization of therapeutic antibodies through traditional techniques, such as candidate screening via hybridoma or phage display, is resource-intensive and time-consuming. In recent years, computational and artificial intelligence-based methods have been actively developed to accelerate and improve the development of therapeutic antibodies. In this study, we developed an end-to-end sequence-based deep learning model, termed AttABseq, for the predictions of the antigen-antibody binding affinity changes connected with antibody mutations. AttABseq is a highly efficient and generic attention-based model by utilizing diverse antigen-antibody complex sequences as the input to predict the binding affinity changes of residue mutations. The assessment on the three benchmark datasets illustrates that AttABseq is 120% more accurate than other sequence-based models in terms of the Pearson correlation coefficient between the predicted and experimental binding affinity changes. Moreover, AttABseq also either outperforms or competes favorably with the structure-based approaches. Furthermore, AttABseq consistently demonstrates robust predictive capabilities across a diverse array of conditions, underscoring its remarkable capacity for generalization across a wide spectrum of antigen-antibody complexes. It imposes no constraints on the quantity of altered residues, rendering it particularly applicable in scenarios where crystallographic structures remain unavailable. The attention-based interpretability analysis indicates that the causal effects of point mutations on antibody-antigen binding affinity changes can be visualized at the residue level, which might assist automated antibody sequence optimization. We believe that AttABseq provides a fiercely competitive answer to therapeutic antibody optimization.

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.

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.

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.

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.

Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting.

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

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.

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.

Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex.

Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.

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.

A link between circulating immune complexes and acute kidney injury in human visceral leishmaniasis.

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum. Clinically, VL evolves with systemic impairment, immunosuppression and hyperactivation with hypergammaglobulinemia. Although renal involvement has been recognized, a dearth of understanding about the underlying mechanisms driving acute kidney injury (AKI) in VL remains. We aimed to evaluate the involvement of immunoglobulins (Igs) and immune complexes (CIC) in the occurrence of AKI in VL patients. Fourteen VL patients were evaluated between early treatment and 12 months post-treatment (mpt). Anti-Leishmania Igs, CIC, cystatin C, C3a and C5a were assessed and correlated with AKI markers. Interestingly, high levels of CIC were observed in VL patients up to 6 mpt. Concomitantly, twelve patients met the criteria for AKI, while high levels of cystatin C were observed up to 6 mpt. Plasmatic cystatin C was positively correlated with CIC and Igs. Moreover, C5a was correlated with cystatin C, CIC and Igs. We did not identify any correlation between amphotericin B use and kidney function markers in VL patients, although this association needs to be further explored in subsequent studies. Our data reinforce the presence of an important renal function impairment during VL, suggesting the involvement of Igs, CIC, and C5a in this clinical condition.

Biomimetic immobilization of α-glucosidase inspired by antibody-antigen specific recognition for catalytic preparation of 4-methylumbelliferone.

Immobilization technology plays an important role in enhancing enzyme stability and environmental adaptability. Despite its rapid development, this technology still encounters many challenges such as enzyme leakage, difficulties in large-scale implementation, and limited reusability. Drawing inspiration from natural paired molecules, this study aimed to establish a method for immobilized α-glucosidase using artificial antibody-antigen interaction. The proposed method consists of three main parts: synthesis of artificial antibodies, synthesis of artificial antigens, and assembly of the artificial antibody-antigen complex. The critical step in this method involves selecting a pair of structurally similar compounds: catechol as a template for preparing artificial antibodies and protocatechualdehyde for modifying the enzyme to create the artificial antigens. By utilizing the same functional groups in these compounds, specific recognition of the antigen by the artificial antibody can be achieved, thereby immobilizing the enzymes. The results demonstrated that the immobilization amount, specific activity, and enzyme activity of the immobilized α-glucosidase were 25.09 ± 0.10 mg/g, 5.71 ± 0.17 U/mgprotein and 143.25 ± 1.71 U/gcarrier, respectively. The immobilized α-glucosidase not only exhibited excellent reusability but also demonstrated remarkable performance in catalyzing the hydrolysis of 4-methylumbelliferyl-α-D-glucopyranoside.

Hydralazine use can be associated with IgM-dominated immune complex-mediated glomerulonephritis.

CONTEXT: IgM-dominant immune complex-mediated glomerulonephritis (IgM-dominant ICMGN) is a rare renal entity, characterized by a membranoproliferative pattern by light microscopy, dominant IgM staining by immunofluorescent staining, and subendothelial deposits by electron microscopy. This study was to investigate if some of IgM-ICMGN were associated with autoimmune disorders induced by hydralazine. DESIGN: Seven IgM-dominant ICMGN cases were identified over 8 years. Their pathologic phenotypes and clinical scenarios were analyzed in detail. RESULTS: Patients' ages ranged from 47 to 87 years old with 5 women and two men. Six of seven patients had drug-induced autoimmune phenomenon (hydralazine-induced positive ANCA and ANA). All of them had renal dysfunction and some proteinuria. Most pathologic features showed a membranoproliferative pattern of glomerulonephritis with dominant IgM deposits at subendothelial spaces. IgM nephropathy (a variant of focal segmental glomerulosclerosis), chronic thrombotic microangiopathy, and cryoglobulinemic glomerulopathy were ruled out in the cases. CONCLUSION: The hydralazine-induced autoimmune phenomenon can be seen in IgM-dominant ICMGN, which should be classified as a subtype of membranoproliferative glomerulonephritis.

Mendelian Causes of Autoimmunity: the Lupus Phenotype.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by its large heterogeneity in terms of clinical presentation and severity. The pathophysiology of SLE involves an aberrant autoimmune response against various tissues, an excess of apoptotic bodies, and an overproduction of type-I interferon. The genetic contribution to the disease is supported by studies of monozygotic twins, familial clustering, and genome-wide association studies (GWAS) that have identified numerous risk loci. In the early 70s, complement deficiencies led to the description of familial forms of SLE caused by a single gene defect. High-throughput sequencing has recently identified an increasing number of monogenic defects associated with lupus, shaping the concept of monogenic lupus and enhancing our insights into immune tolerance mechanisms. Monogenic lupus (moSLE) should be suspected in patients with either early-onset lupus or syndromic lupus, in male, or in familial cases of lupus. This review discusses the genetic basis of monogenic SLE and proposes its classification based on disrupted pathways. These pathways include defects in the clearance of apoptotic cells or immune complexes, interferonopathies, JAK-STATopathies, TLRopathies, and T and B cell dysregulations.

Leaky gut, circulating immune complexes, arthralgia, and arthritis in IBD: coincidence or inevitability?

Most host-microbiota interactions occur within the intestinal barrier, which is essential for separating the intestinal epithelium from toxins, microorganisms, and antigens in the gut lumen. Gut inflammation allows pathogenic bacteria to enter the blood stream, forming immune complexes which may deposit on organs. Despite increased circulating immune complexes (CICs) in patients with inflammatory bowel disease (IBD) and discussions among IBD experts regarding their potential pathogenic role in extra-intestinal manifestations, this phenomenon is overlooked because definitive evidence demonstrating CIC-induced extra-intestinal manifestations in IBD animal models is lacking. However, clinical observations of elevated CICs in newly diagnosed, untreated patients with IBD have reignited research into their potential pathogenic implications. Musculoskeletal symptoms are the most prevalent extra-intestinal IBD manifestations. CICs are pivotal in various arthritis forms, including reactive, rheumatoid, and Lyme arthritis and systemic lupus erythematosus. Research indicates that intestinal barrier restoration during the pre-phase of arthritis could inhibit arthritis development. In the absence of animal models supporting extra-intestinal IBD manifestations, this paper aims to comprehensively explore the relationship between CICs and arthritis onset via a multifaceted analysis to offer a fresh perspective for further investigation and provide novel insights into the interplay between CICs and arthritis development in IBD.

Interleukin-2/anti-interleukin-2 immune complex attenuates cold ischemia-reperfusion injury after kidney transplantation by increasing renal regulatory T cells.

BACKGROUND: Cold ischemia-reperfusion injury (IRI) is an unavoidable complication of kidney transplantation. We investigated the role of regulatory T cells (Treg) in cold IRI and whether the interleukin (IL)-2/anti-IL-2 antibody complex (IL-2C) can ameliorate cold IRI. METHODS: We developed a cold IRI mouse model using kidney transplantation and analyzed the IL-2C impact on cold IRI in acute, subacute and chronic phases. RESULTS: Treg transfer attenuated cold IRI, while Treg depletion aggravated cold IRI. Next, IL-2C administration prior to IRI mitigated acute renal function decline, renal tissue damage and apoptosis and inhibited infiltration of effector cells into kidneys and pro-inflammatory cytokine expression on day 1 after IRI. On day 7 after IRI, IL-2C promoted renal regeneration and reduced subacute renal damage. Furthermore, on day 28 following IRI, IL-2C inhibited chronic fibrosis. IL-2C decreased reactive oxygen species-mediated injury and improved antioxidant function. When IL-2C was administered following IRI, it also increased renal regeneration with Treg infiltration and suppressed renal fibrosis. In contrast, Treg depletion in the presence of IL-2C eliminated the positive effects of IL-2C on IRI. CONCLUSION: Tregs protect kidneys from cold IRI and IL-2C inhibited cold IRI by increasing the renal Tregs, suggesting a potential of IL-2C in treating cold IRI. KEY POINTS: Interleukin (IL)-2/anti-IL-2 antibody complex attenuated acute renal injury, facilitated subacute renal regeneration and suppressed chronic renal fibrosis after cold ischemia-reperfusion injury (IRI) by increasing the renal Tregs. IL-2/anti-IL-2 antibody complex decreased reactive oxygen species-mediated injury and improved antioxidant function. This study suggests the therapeutic potential of the IL-2/anti-IL-2 antibody complex in kidney transplantation-associated cold IR.