PV116 / #181 Poster Topic:AS14 - Innate Immunity Complement plays a central role in SLE, generating an array of bioactive soluble and cell-bound complement activation products (CB-CAPs) during disease activity. Data are lacking though detailing the types, quantities, and impacts of the numerous CB-CAP on SLE immune cells, especially with respect to disease activity. We applied a mass cytometry (MC) panel that can detect over 20 CB-CAPs and complement receptors to PBMCs from paired flare and remission samples from 6 patients with classified SLE. Furthermore, we analyzed single-cell transcriptional profiles on flaring samples using antibodies to the most prevalent CB-CAPs using cellular indexing of transcriptomes and epitopes (CITE)-seq. Adults with ACR- or SLICC-classified SLE were consented for PBMC collection at Washington University School of Medicine. Isolated PBMCs were subjected to single-cell MC (n = 6) and CITE-seq (n = 3). MC data analysis was performed with Cytobank. CITE-seq data analyses was performed with Seuret, gProfileR, and Comprehensive Multi-omics Platform for Biological InterpretatiOn (COMPBIO). We found the highest frequency of C4d, C3d, C5, and Bb deposition on B cells compared to T cells and monocytes during SLE flares (Figure 1). During disease remission, low levels of all CB-CAPs were observed in these cells. Compared to controls, transitional B cells from flaring patients with SLE had high levels of C5 and Bb with little C4d or C3d. CD11c+B cells from flaring patients also had elevated Bb deposition compared to controls. CITE-seq transcriptional profiling identified Bb- and C3d-bearing CD11c+B cells possessing a type I interferon signature, with Bb-bearing B cells further possessing a TNF/NF-κB transcriptional signature (Figure 2). Figure 1. Figure 2. A high level of CB-CAP deposition was observed in B cells obtained from flaring subjects with a SLE, which was absent during disease remission. The types of CB-CAPs found on PBMCs were not uniform between cell types, potentially opening a previously undescribed heterogeneity in SLE. Additional heterogeneity was observed in the transcriptional profiles associated with specific CB-CAPs on B cells. These pilot data demonstrate the feasibility of the MC complement panel on human samples, and the potential insights CITE-seq has using CB-CAPs in discovering novel mechanisms of complement activation and regulation.

BACKGROUND:During the COVID-19 pandemic, novel nanoparticle-based mRNA vaccines were developed. A small number of individuals developed allergic reactions to these vaccines although the mechanisms remain undefined. METHODS:To understand COVID-19 vaccine-mediated allergic reactions, we enrolled 19 participants who developed allergic events within 2 h of vaccination and 13 controls, nonreactors. Using standard hemolysis assays, we demonstrated that sera from allergic participants induced stronger complement activation compared to nonallergic subjects following ex vivo vaccine exposure. RESULTS:Vaccine-mediated complement activation correlated with anti-polyethelyne glycol (PEG) IgG (but not IgM) levels while anti-PEG IgE was undetectable in all subjects. Depletion of total IgG suppressed complement activation in select individuals. To investigate the effects of vaccine excipients on basophil function, we employed a validated indirect basophil activation test that stratified the allergic populations into high and low responders. Complement C3a and C5a receptor blockade in this system suppressed basophil response, providing strong evidence for complement involvement in vaccine-mediated basophil activation. Single-cell multiome analysis revealed differential expression of genes encoding the cytokine response and Toll-like receptor (TLR) pathways within the monocyte compartment. Differential chromatin accessibility for IL-13 and IL-1B genes was found in allergic and nonallergic participants, suggesting that in vivo, epigenetic modulation of mononuclear phagocyte immunophenotypes determines their subsequent functional responsiveness, contributing to the overall physiologic manifestation of vaccine reactions. CONCLUSION:These findings provide insights into the mechanisms underlying allergic reactions to COVID-19 mRNA vaccines, which may be used for future vaccine strategies in individuals with prior history of allergies or reactions and reduce vaccine hesitancy.

Complement is a major innate defense system that protects the intravascular space from microbial invasion. Complement activation results in the assembly of C3 convertases, serine proteases that cleave complement protein C3, generating bioactive fragments C3a and C3b. The complement response is rapid and robust, largely due to a positive feedback regulatory loop mediated by alternative pathway (AP) C3 convertase. C3 nephritic factors (C3NEFs) are autoantibodies that stabilize AP convertase, resulting in uncontrolled C3 cleavage, which, in principle, can promote critical tissue injury similar to that seen in certain renal conditions. Investigations of C3NEFs are hampered by a challenging issue: each C3NEF is derived from a different donor source, and there is no method to compare one C3NEF to another. We have identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in a form resistant to decay acceleration by multiple complement regulators. The antibody requires the presence of properdin to confer convertase stability, and hampers the activity of Salp20, a tic salivary protein that accelerates convertase dissociation by displacing properdin from the convertase complex. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

Objective To characterize lesion evolution and neurodegeneration in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) using multimodal MRI. Methods We prospectively performed MRI and cognitive testing in RVCL-S and healthy control cohorts. Gray and white matter volume and disruption of white matter microstructure were quantified. Asymmetric spin echo acquisition permitted voxel-wise oxygen extraction fraction (OEF) calculation as an in vivo marker of microvascular ischemia. The RVCL-S cohort was included in a longitudinal analysis of lesion subtypes in which hyperintense lesions on fluid-attenuated inversion recovery (FLAIR), T1-postgadolinium, and diffusion-weighted imaging were delineated and quantified volumetrically. Results Twenty individuals with RVCL-S and 26 controls were enrolled. White matter volume and microstructure declined faster in those with RVCL-S compared to controls. White matter atrophy in RVCL-S was highly linear (rho = -0.908, p < 0.0001). Normalized OEF was elevated in RVCL-S and increased with disease duration. Multiple cognitive domains, specifically those measuring working memory and processing speed, were impaired in RVCL-S. Lesion volumes, regardless of subtype, progressed/regressed with high variability as a function of age, while FLAIR lesion burden increased near time to death (p < 0.001). Conclusion RVCL-S is a monogenic microvasculopathy affecting predominantly the white matter with regard to atrophy and cognitive impairment. White matter volumes in RVCL-S declined linearly, providing a potential metric against which to test the efficacy of future therapies. Progressive elevation of white matter OEF suggests that microvascular ischemia may underlie neurodegeneration in RVCL-S.

Objective To characterize lesion evolution and neurodegeneration in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) using multimodal MRI. Methods We prospectively performed MRI and cognitive testing in RVCL-S and healthy control cohorts. Gray and white matter volume and disruption of white matter microstructure were quantified. Asymmetric spin echo acquisition permitted voxel-wise oxygen extraction fraction (OEF) calculation as an in vivo marker of microvascular ischemia. The RVCL-S cohort was included in a longitudinal analysis of lesion subtypes in which hyperintense lesions on fluid-attenuated inversion recovery (FLAIR), T1-postgadolinium, and diffusion-weighted imaging were delineated and quantified volumetrically. Results Twenty individuals with RVCL-S and 26 controls were enrolled. White matter volume and microstructure declined faster in those with RVCL–S compared to controls. White matter atrophy in RVCL-S was highly linear (ρ = −0.908, p < 0.0001). Normalized OEF was elevated in RVCL-S and increased with disease duration. Multiple cognitive domains, specifically those measuring working memory and processing speed, were impaired in RVCL-S. Lesion volumes, regardless of subtype, progressed/regressed with high variability as a function of age, while FLAIR lesion burden increased near time to death (p < 0.001). Conclusion RVCL-S is a monogenic microvasculopathy affecting predominantly the white matter with regard to atrophy and cognitive impairment. White matter volumes in RVCL-S declined linearly, providing a potential metric against which to test the efficacy of future therapies. Progressive elevation of white matter OEF suggests that microvascular ischemia may underlie neurodegeneration in RVCL-S.

CD4+T-helper 1 cell(Th1)function is closely regulated by an intrinsic developmental program in which activation/induction and proinflammatory interferon(IFN)-y secretion are followed by a deactivation/contraction period characterized by a switch into cosecretion of immunoregulatory interleukin(IL)-10.Autocrine intracellular complement(complosome)activity plays a vital role in Th1 initiation and contraction:T-cell receptor(TCR)stimulation induces intracellular activation of the key complement compo-nents C3(through cathepsin L(CTSL)cleavage)and C5,which leads to intrinsic engagement of CD46 by C3b,of the C3a receptor(C3aR)by C3a,and of the C5a receptors(C5aR1 and C5aR2)by C5a.1,2 These events mediate the metabolic programming required for IFN-y production and Th1 induction.3 CD46-mediated signals also support subsequent IL-10 switching and Th1 contraction by increasing the oxidative phosphorylation to glycolysis ratio,while autocrine C5aR2 engagement by secreted des-arginated C5a(C5a-desArg)suppresses intracellular C5aR1 activity(Supplementary Fig.1a depicts a model summarizing the role of the complosome in Th1 induction and contraction).

Background Complement plays a central role in SLE. Its activation by immune complexes drives type II and III hypersensitivity reactions and classical pathway activation, leading to inflammatory responses in target tissues. Its activation generates an array of bioactive soluble and cell-bound complement activation products (CB-CAPs). Data are lacking detailing the types and quantities of each CB-CAP on SLE immune cells. We speculate that discerning the patterns and impacts of complement signatures on SLE B cells will provide insight into the role of complement activation in the pathogenesis of SLE. To characterize the array of CB-CAPs on SLE immune cells, we created and validated a mass cytometry (MC) panel suitable for detecting over 20 CB-CAPs and complement receptors, then examined PBMCs from paired flare and inactive samples from patients with SLE. Methods Adults with ACR- or SLICC-classified SLE (n=5) were enrolled and consented for PBMC collection at Washington University School of Medicine. Paired samples were obtained, one during a flare and one after resolution of the flare. Frozen ficoll gradient isolated buffy coat PBMCs were thawed counted, filtered, and treated with cisplatin to confirm viability. Prepared cells were stained with metal-conjugated antibodies to measure surface complement proteins and markers identifying immune cell subset. Stained cells were treated with an Iridium intercalator stain, recounted, washed in Milli-Q water to remove salts, resuspended with a solution of bead standards to facilitate data normalization, and subjected to single cell MC. Sample to sample consistency is optimized by simultaneously examining multiple cell preparations barcoded with palladium metals. Data analysis was performed with Cytobank. Distinct cell sub-populations were identified and organized in a hierarchal fashion. Results We found the highest frequency of C4d deposition (indicative of classical pathway activation) on B cells compared to T cells and monocytes during SLE flares. Surprising, only B cells had C3d, C5 (common pathway), and Bb (alternative pathway) CAPs with none observed on T cells or monocytes suggesting that incomplete classical pathway activation occurred on these cells. We also found that different B cell subsets differentially activated complement during flares. Transitional B cells universally had C4d and C5 but surprisingly had little C3d, despite the presence of Bb. Mature B cells, particularly the mature anergic subset, and memory B cells had C4d and C3d but little C5. Plasma cells/plasmablasts were devoid of any CB-CAPs. Finally, during disease inactivity, very low levels of all CB-CAPs were observed on all PBMCs. Conclusions We found a high level of CB-CAP deposition in immune cells obtained from subjects with a SLE flare, which was absent during disease inactivity. The types of CB-CAPs on PBMCs were not uniform, and our data suggest that certain immune cells appear regulate complement activation while others do not. The reasons for this are unclear, but it potentially opens up a previously undescribed heterogeneity in SLE. These pilot data demonstrate the feasibility of the MC complement panel on human samples and the power of this approach in discovering novel mechanisms of complement activation and regulation. Funding Source(s): AHK: NIH/NIAMS R21 AR069833, Doris Duke Charitable Foundation

Neutrophils are essential to the pathogenesis of many inflammatory diseases. In the autoantibody-mediated K/BxN model of inflammatory arthritis, the alternative pathway (AP) of complement and Fc gamma receptors (FcγRs) are required for disease development while the classical pathway is dispensable. The reason for this differential requirement is unknown. We show that within minutes of K/BxN serum injection complement activation (CA) is detected on circulating neutrophils, as evidenced by cell surface C3 fragment deposition. CA requires the AP factor B and FcγRs but not C4, implying that engagement of FcγRs by autoantibody or immune complexes directly triggers AP C3 convertase assembly. The absence of C5 does not prevent CA on neutrophils but diminishes the upregulation of adhesion molecules. In vivo two-photon microscopy reveals that CA on neutrophils is critical for neutrophil extravasation and generation of C5a at the site of inflammation. C5a stimulates the release of neutrophil proteases, which contribute to the degradation of VE-cadherin, an adherens junction protein that regulates endothelial barrier integrity. C5a receptor antagonism blocks the extracellular release of neutrophil proteases, suppressing VE-cadherin degradation and neutrophil transendothelial migration in vivo. These results elucidate the AP-dependent intravascular neutrophil-endothelial interactions that initiate the inflammatory cascade in this disease model but may be generalizable to neutrophil extravasation in other inflammatory processes.

ProblemCrry is a widely expressed type 1 transmembrane complement regulatory protein in rodents which protects self‐tissue by downregulating C3 activation. Crry−/− concepti produced by Crry+/− × Crry+/− matings are attacked by maternal complement system leading to loss before day 10. The membrane attack complex is not the mediator of this death. We hypothesized that the ability of C3b to engage the alternative pathway's feedback loop relatively unchecked on placental membranes induces the lesion yielding the demise of the Crry−/− mouse.Method of StudyWe investigated the basis of Crry−/− conceptus demise by depleting maternal complement with cobra venom factor and blocking antibodies. We monitored their effects primarily by genotyping and histologic analyses.ResultsWe narrowed the critical period of the complement effect from 6.5 to 8.5 days post‐coitus (dpc), which is immediately after the conceptus is exposed to maternal blood. Deposition by 5.5 dpc of maternal C3b on the placental vasculature lacking Crry−/− yielded loss of the conceptus by 8.5 dpc. Fusion of the allantois to the chorion during placental assembly did not occur, fetal vessels originating in the allantois did not infiltrate the chorioallantoic placenta, the chorionic plate failed to develop, and the labyrinthine component of the placenta did not mature.ConclusionOur data are most consistent with the deposition of C3b being responsible for the failure of the allantois to fuse to the chorion leading to subsequent conceptus demise.

The complexes trans-[RuCl2(dppb)(cydn)] (1), trans-[RuCl2(dppb)(opda)] (2) and cis-[RuCl2(dppb)(cydn)] (3) were synthesized from [{RuCl2(dppb)}2-μ-(dppb)] {where: dppb = 1,4-bis(diphenylphosphino)butane; cydn = cis and trans (±) 1,2-diaminocyclohexane, and opda = o-phenylenediamine}. The complexes were characterized by nuclear magnetic resonance of phosphorus (31P{1H} NMR), cyclic voltammetry (CV), infrared and ultraviolet/visible spectra (IR and UV/vis) as well as elemental analyses (CHN). The X-ray structures of (1) and (3) were determined and they are presented here. DFT calculations and experimental data showed that the trans isomers are obtained as thermodynamic products while the cis isomers are kinetic products. This behavior is different than described in the literature for similar complexes, where the cis isomer is obtained from the trans isomer. Additionally, the catalytic activity of the complexes (1), (2) and (3) was investigated, as pre-catalysts, in the reduction of the acetophenone and 4-methylacetophenone by transfer-hydrogenation.

During infection and budding, human immunodeficiency virus-1 (HIV-1) acquires regulators of Complement Activation (RCAs) along with the host cell membrane on the viral envelope. Activation of host complement system results in opsonization of virus by complement fragments, however the virus evades complement mediated lysis (CoML) by virtue of the RCAs on the viral envelope. The RCAs on HIV-1 envelope process complement protein C3 into various fragments that promote viral entry and infection of cells through different complement receptors. Complement opsonized HIV-1 has been shown in vitro to infect dendritic cells (DCs) in a CR3 dependent manner, although the role of CR3 and CD46 in natural HIV-1 infection is not clear. Surface expression of CR3 and CD46 on DC subsets of 30 antiretroviral naïve, 31 treated (cART) HIV-1 infected individuals and 30 seronegative controls was measured by flow cytometry and plasma levels of cytokines and complement activity (C3c levels) were quantitated by sandwich ELISA. Significantly lower surface expression of CR3 and CD46 was observed on DC subsets in naïve and treated HIV-1 infected individuals compared to controls. Significantly higher complement activation and plasma levels of IL-4, IL-8, IL-10 and IFN-γ were observed in treatment naïve HIV-1 infected individuals than controls. Significantly lower plasma levels of IL-4, IL-6, IL-8 and IL-10 were observed in treated vs. naïve HIV-1 infected individuals. Our findings suggest that alterations in expression of CR3 and CD46 on DCs along with complement activity could be factors that influence viral persistence and HIV-1 disease progression and need to be further evaluated.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide with elusive molecular mechanisms. The aim of this study is to investigate the clinical significance and biological roles of breast cancer-associated protein 3 (BCA3) in HCC. Our investigation demonstrated that BCA3 expression was up-regulated in primary HCC tissues, and BCA3 levels were positively correlated with tumor size, TNM stage, microvascular invasion and poor prognosis. BCA3 promoted tumor growth, metastasis and angiogenesis of HCC in vitro and in vivo. Moreover, we found that BCA3 induced aggressive behaviors were mediated by AKT activation, which in turn activated mTOR signalling pathway and induced cytoplasm-nuclear translocation of NF-κB p65. Blockage of AKT signalling pathway by a specific AKT inhibitor LY294002 impaired BCA3 mediated phenotypes. Collectively, our current study indicated the pleiotropic effects of BCA3 in HCC progression, and blockage of BCA3-AKT pathway might contribute to development of therapeutic measures for HCC.

Factor D (FD) is an essential component of the complement alternative pathway (AP). It is an attractive pharmaceutical target because it is an AP-specific protease circulating in blood. Most components of the complement activation pathways are produced by the liver, but FD is highly expressed by adipose tissue. Two critical questions are: 1) to what degree does adipose tissue contribute to circulating FD levels and 2) what quantity of FD is sufficient to maintain a functional AP? To address these issues, we studied a novel mouse strain with complete lipodystrophy (LD), the fld mouse with partial LD, an FD-deficient mouse, and samples from lipodystrophic patients. FD was undetectable in the serum of LD mice, which also showed minimal AP function. Reconstitution with purified FD, serum mixing experiments, and studies of partial LD mice all demonstrated that a low level of serum FD is sufficient for normal AP activity in the mouse system. This conclusion was further supported by experiments in which wild-type adipose precursors were transplanted into LD mice. Our results indicate that almost all FD in mouse serum is derived from adipose tissue. In contrast, FD levels were reduced ∼50% in the sera of patients with congenital generalized LD. Our studies further demonstrate that a relatively small amount of serum FD is sufficient to facilitate significant time-dependent AP activity in humans and in mice. Furthermore, this observation highlights the potential importance of obtaining nearly complete inhibition of FD in treating alternative complement activation in various autoimmune and inflammatory human diseases.