Effects Of Complement Activation Research Articles

Exploring the dual role of B cells in solid tumors: implications for head and neck squamous cell carcinoma.

In the tumor milieu of head and neck squamous cell carcinoma (HNSCC), distinct B cell subpopulations are present, which exert either pro- or anti-tumor activities. Multiple factors, including hypoxia, cytokines, interactions with tumor cells, and other immune infiltrating lymphocytes (TILs), alter the equilibrium between the dual roles of B cells leading to cancerogenesis. Certain B cell subsets in the tumor microenvironment (TME) exhibit immunosuppressive function. These cells are known as regulatory B (Breg) cells. Breg cells suppress immune responses by secreting a series of immunosuppressive cytokines, including IL-10, IL-35, TGF-β, granzyme B, and adenosine or dampen effector TILs by intercellular contacts. Multiple Breg phenotypes have been discovered in human and mouse cancer models. However, when compartmentalized within a tertiary lymphoid structure (TLS), B cells predominantly play anti-tumor effects. A mature TLS contains a CD20+ B cell zone with several important types of B cells, including germinal-center like B cells, antibody-secreting plasma cells, and memory B cells. They kill tumor cells via antibody-dependent cytotoxicity and phagocytosis, and local complement activation effects. TLSs are also privileged sites for local T and B cell coordination and activation. Nonetheless, in some cases, TLSs may serve as a niche for hidden tumor cells and indicate a bad prognosis. Thus, TIL-B cells exhibit bidirectional immune-modulatory activity and are responsive to a variety of immunotherapies. In this review, we discuss the functional distinctions between immunosuppressive Breg cells and immunogenic effector B cells that mature within TLSs with the focus on tumors of HNSCC patients. Additionally, we review contemporary immunotherapies that aim to target TIL-B cells. For the development of innovative therapeutic approaches to complement T-cell-based immunotherapy, a full understanding of either effector B cells or Breg cells is necessary.

Abstract 154: Crossroads Of Immunity And Coagulation: Protease Activity Of Complement Component C1s Inhibits Protein C Activation In Covid-19

Introduction: There is an abundance of crosstalk between innate immunity and coagulation in thrombo-inflammatory diseases such as COVID-19. Several potential procoagulant effects of complement activation have been described, but whether complement proteases affect antithrombotic pathways in is not known. Aims: We tested the hypothesis that complement component 1 (C1) impairs the protein C antithrombotic pathway. Methods: Plasma levels of the C1 complex were measured in plasma collected from healthy individuals and COVID-19 patients hospitalized early in the COVID-19 pandemic (NCT04360824). Activation of protein C by recombinant thrombomodulin was measured in a two-stage assay. In the first stage, protein C, thrombin, and thrombomodulin were incubated to generate activated protein C. The reaction mix was incubated with either healthy or COVID-19 plasma in the presence or absence of C1 inhibitor (which binds to C1 to inhibit its activity) or sutimlimab (a specific C1s inhibitor). During the second stage, protein C activation was measured using spectrophotometry with the chromogenic substrate S-2366. Results: In the inflammatory condition COVID-19, plasma levels of the C1 complex were increased compared to healthy controls ( p = 0.004). Generation of activated protein C by recombinant thrombomodulin was decreased in the presence of COVID-19 plasma ( p = 0.001), whereas the addition of healthy plasma had no significant effect ( p = 0.469). Addition of C1 inhibitor or sutimlimab normalized protein C activation in the presence of COVID-19 plasma ( p = 0.022 and 0.039, respectively). Addition of recombinant C1s protease, but not C1r protease or C1q, impaired the generation of activated protein C in a dose dependent fashion. Conclusions: We have uncovered a novel role of the protease C1s in regulating the generation of anticoagulant activated protein C. These findings suggest that the classical pathway of complement activation may impair anticoagulant responses in COVID-19.

Upregulation of complement proteins in lung cancer cells mediates tumor progression.

In vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture. Studies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq. Changes in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner. Based on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing.

Complement complex 1 subunit q-mediated hepatic stellate cell activation with connective tissue growth factor elevation is a prognostic factor for survival in rat and human chronic liver diseases.

Complement complex 1 subunit q (C1q) has multiple functions, including cell migration, in addition to its traditional complement-activating effect. Research shows C1q is a ligand for frizzled receptors (FZDs). FZD-induced yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) alternate Wnt signaling activation induces connective tissue growth factor (CTGF) production and hepatic stellate cell (HSC) activation. However, no study exists in which C1q directly induces CTGF in HSCs. Here, we investigated the role of C1q in HSC activation. Human HSCs (LX2) were incubated with C1q to assess HSC activation. C1q and fibrotic markers were assessed using immunohistochemistry, immunoblotting, and quantitative reverse-transcription polymerase chain reaction in cirrhotic rats administered CCl4 for 21 weeks. Serum C1q, liver function, and fibrosis score were measured in 91 patients with chronic liver disease. The correlations between serum C1q and liver function, fibrosis score, and survival prognosis were examined. C1q-activated LX2s showed morphologic changes, up-regulation of CTGF, tissue inhibitors of metalloproteinases (TIMP-1),and alternate Wnt signal genes FZD2, TAZ, and cysteine-rich angiogenic inducer 61 (Cyr61). Cirrhotic rat liver C1q expression correlated with the Azan-positive area and expression of CTGF, TIMP-1, hyaluronan synthase (HAS)1, HAS3, and CD44. Expression of C1q protein and C1q, CTGF, and TIMP-1 genes were higher in deceased cirrhotic rat livers compared to surviving rats. Human serum C1q levels increased in liver cirrhosis compared to chronic hepatitis and correlated with liver fibrosis and functional markers. Ten patients suffered liver-related death over a 66-month observation period. The C1q cut-off value (11 mg/dl) showed patients with serum values < 11 mg/dl had longer rates of survival compared to C1q ≥ 11 mg/dl. Conclusion: C1q-mediated HSC activation in liver fibrosis is associated with CTGF elevation. Additionally, serum C1q may be diagnostic for survival in human chronic liver diseases.

Complement Is Activated During Normothermic Machine Perfusion of Porcine and Human Discarded Kidneys.

BackgroundThe gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP.MethodsNMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition.ResultsComplement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death.ConclusionNormothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation.

Increased cell stiffness contributes to complement-mediated injury of choroidal endothelial cells in a monkey model of early age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet no therapies exist for ~85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigmented epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of the small GTPases Rac and Rho, because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility, while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential. © 2022 The Pathological Society of Great Britain and Ireland.

Complement-mediated release of fibroblast growth factor 2 from human RPE cells

PurposeComplement activation is associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). Fibroblast growth factor 2 (FGF2) and membrane attack complex (MAC) are present in eyes of patients with CNV. Herein, we investigated the effect of complement activation on FGF2 release in human retinal pigment epithelial (RPE) cells. MethodsCultured human RPE cells were primed with an anti-RPE antibody and then treated with C1q-depleted human serum in the presence or absence of Tec kinases inhibitor (LFM-A13). 38 cytokines/chemokines levels were measured by Luminex technology. Secretion of FGF2 and interleukin (IL)-6 was assessed by ELISA. Tec protein was measured by Western blot. mRNA expression of FGF2, chemokine (C-X-C motif) ligand 1 (CXCL-1), and family members of Tec kinases was evaluated by qPCR. Cell viability and MAC deposition were determined by WST-1 assay and flow cytometry, respectively. ResultsComplement activation caused increased FGF2 and IL-6 release. FGF2 was released when C6-depleted human serum was reconstituted with C6. Anti-C5 antibody significantly attenuated complement-mediated FGF2 release, but not IL-6. FGF2 mRNA levels were not affected, while CXCL-1 mRNA levels were increased by complement activation. FGF2-containing extracellular vesicles were detected in response to complement challenge. Tec mRNA and protein were expressed in RPE cells. In the presence of LFM-A13, secretion of FGF2, but not IL-6, and MAC deposition were significantly decreased and cell viability was significantly increased in complement-treated cells when compared to controls. ConclusionsComplement plays an important role to release FGF2 from RPE cells. Tec kinase is involved in MAC formation and complement-mediated FGF2 release. This information suggests a role for complement activation to mediate neovascularization in conditions such as AMD, and may elucidate potential therapeutic targets.

The complement system in COVID-19: friend and foe?

Coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in a global pandemic and a disruptive health crisis. COVID-19-related morbidity and mortality have been attributed to an exaggerated immune response. The role of complement activation and its contribution to illness severity is being increasingly recognized. Here, we summarize current knowledge about the interaction of coronaviruses with the complement system. We posit that (a) coronaviruses activate multiple complement pathways; (b) severe COVID-19 clinical features often resemble complementopathies; (c) the combined effects of complement activation, dysregulated neutrophilia, endothelial injury, and hypercoagulability appear to be intertwined to drive the severe features of COVID-19; (d) a subset of patients with COVID-19 may have a genetic predisposition associated with complement dysregulation; and (e) these observations create a basis for clinical trials of complement inhibitors in life-threatening illness.

Formation of Protein Corona on Nanoparticle Affects Different Complement Activation Pathways Mediated by C1q.

As nanoparticles (NPs) are intravenously entering the bloodstream, proteins in the plasma can recognize and bind them to form a protein corona that affects how NPs are perceived by biological systems. The complement is an essential part of the innate immunity that contributes to non-specific host defense. How complement recognizes NPs has not been elucidated. Here, we developed a proteomics and biochemical approach to understand the applied risk of activated complement by NPs. Complement proteins absorbed on Hydroxyapatite Nanoparticles (HAP-NPs) and Silicon dioxide Nanoparticles (SiO2-NPs) were analyzed by proteomics with LC-MS. The effect of complement activation was studied by iC3b/Sc5b-9/C3a/C4a/C5a with ELISA. An inhibitory model was established via EDTA and EGTA to confirm the selective pathway activation of both NPs. Finally, the regulation of complement by NPs was analyzed by western blot. The results indicate that HAP-NPs start the activation of the complement through the classical pathway because of the absorption of C1q and the release of C1r/C1s. Meanwhile, the soluble regulatory molecules such as CFI, C4bp, and CFH tried to resist the complement system activation by the cleavage of C3 convertase. In contrast, SiO2-NPs can activate the alternative pathway of the complement through the absorption of CFD and CFB. It was clarified that HAP-NPs and SiO2-NPs activate complement through different mechanisms. These studies provide a scientific basis for the design and modification of nano-drug carriers for delaying their recognition and clearance by the mononuclear phagocytic system and simultaneously reducing the immunotoxicity of NPs. The understanding of protein corona is conducive to innovation in the field of "immune-safe-by-design" nanomedicines.

The complex functioning of the complement system in xenotransplantation.

The role of complement in xenotransplantation is well-known and is a topic that has been reviewed previously. However, our understanding of the immense complexity of its interaction with other constituents of the innate immune response and of the coagulation, adaptive immune, and inflammatory responses to a xenograft is steadily increasing. In addition, the complement system plays a function in metabolism and homeostasis. New reviews at intervals are therefore clearly warranted. The pathways of complement activation, the function of the complement system, and the interaction between complement and coagulation, inflammation, and the adaptive immune system in relation to xenotransplantation are reviewed. Through several different mechanisms, complement activation is a major factor in contributing to xenograft failure. In the organ-source pig, the detrimental influence of the complement system is seen during organ harvest and preservation, for example, in ischemia-reperfusion injury. In the recipient, the effect of complement can be seen through its interaction with the immune, coagulation, and inflammatory responses. Genetic-engineering and other therapeutic methods by which the xenograft can be protected from the effects of complement activation are discussed. The review provides an updated source of reference to this increasingly complex subject.

Upregulation and release of soluble fms-like tyrosine kinase receptor 1 mediated by complement activation in human syncytiotrophoblast cells.

Antiangiogenic molecule soluble fms-like tyrosine kinase receptor 1 (sFLT1) released from trophoblast cells is associated with pregnancy-specific hypertensive disorder pre-eclampsia. Cause of elevated sFLT1 in pre-eclampsia patients is not well understood. Despite evidence of excess systemic and placental complement activation in pre-eclampsia patients, its role in pathophysiology is not clear. If the complement activation plays a role in upregulation and secretion of sFLT1 is not known. Human trophoblast cells were isolated from term placentas and allowed to syncytialize. Complement was activated in vitro at sublethal levels on syncytiotrophoblast cells. Effect of complement activation on expression and release of sFLT1 was assessed by comparing its levels in these cells with and without complement activation. Sublethal level of complement activation on syncytialized human trophoblast cells induced upregulation of sFLT1 mRNA and protein. Complement also induced secretion of sFLT1 in a manner depending on degree of activation. Anaphylatoxins C3a induced upregulation but not the release of sFLT1. Release of terminal membrane attack complex (MAC) was associated with sFLT1 secretion. Complement activation plays a major role in both the expression and secretion of sFLT1 from syncytial trophoblast cells. The terminal MAC complex is involved in its secretion. Increased levels of sFLT1 in pre-eclampsia patients may be due to complement-induced upregulation and secretion.

The Complement System in Dialysis: A Forgotten Story?

Significant advances have lead to a greater understanding of the role of the complement system within nephrology. The success of the first clinically approved complement inhibitor has created renewed appreciation of complement-targeting therapeutics. Several clinical trials are currently underway to evaluate the therapeutic potential of complement inhibition in renal diseases and kidney transplantation. Although, complement has been known to be activated during dialysis for over four decades, this area of research has been neglected in recent years. Despite significant progress in biocompatibility of hemodialysis (HD) membranes and peritoneal dialysis (PD) fluids, complement activation remains an undesired effect and relevant issue. Short-term effects of complement activation include promoting inflammation and coagulation. In addition, long-term complications of dialysis, such as infection, fibrosis and cardiovascular events, are linked to the complement system. These results suggest that interventions targeting the complement system in dialysis could improve biocompatibility, dialysis efficacy, and long-term outcome. Combined with the clinical availability to safely target complement in patients, the question is not if we should inhibit complement in dialysis, but when and how. The purpose of this review is to summarize previous findings and provide a comprehensive overview of the role of the complement system in both HD and PD.

Reduced Expression of Membrane Complement Regulatory Protein CD59 on Leukocytes following Lung Transplantation.

Cellular protection against undesired effects of complement activation is provided by expression of membrane-bound complement regulatory proteins including CD59. This protein prevents membrane attack complex formation and is considered to be involved in graft accommodation. Also, CD59 downregulates CD4+ and CD8+ T-cell activation and proliferation. It is unknown whether CD59 expression is affected by transplantation. The aim of this study was to evaluate the quantitative CD59 antigen expression on distinct leukocyte subsets following lung transplantation (n = 26) and to investigate whether this differs from pretransplantation (n = 9). The results show that CD59 expression on leukocytes is significantly lower posttransplantation compared with healthy controls (p = 0.002) and pretransplantation (p < 0.0001). Moreover, the CD59 expression diminishes posttransplantation on all distinct lymphocyte subsets (p < 0.02). This effect appeared to be specific for CD59 since the expression of other surface markers remained stable or inclined following transplantation. The highest antigen expression posttransplantation was observed on CD4+ T cells and monocytes (p ≤ 0.002). These findings show that CD59 expression on leukocytes diminishes posttransplantation, which could result in decreased resistance against complement and enhanced T-cell activation. If such reduction in CD59 expression also occurs on endothelial cells from the transplanted organ, this could lead to a change into a prothrombotic and proinflammatory phenotype.

Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells.

Purpose. Complement activation is implicated in the pathogenesis of age-related macular degeneration (AMD). Apolipoprotein E (ApoE) and complement activation products such as membrane attack complex (MAC) are present in eyes of individuals with AMD. Herein, we investigated the effect of complement activation on induction of ApoE accumulation in human retinal pigment epithelial (RPE) cells.Methods. Cultured human RPE cells were primed with a complement-fixing antibody followed by treatment with C1q-depleted (C1q-Dep) human serum to elicit alternative pathway complement activation. Controls included anti-C5 antibody-treated serum and heat-inactivated C1q-Dep. Total protein was determined on RPE cell extracts, conditioned media, and extracellular matrix (ECM) by Western blot. ApoE and MAC colocalization was assessed on cultured RPE cells and human eyes by immunofluorescent stain. ApoE mRNA expression was evaluated by quantitative PCR (qPCR).Results. Complement challenge upregulated cell-associated ApoE, but not apolipoprotein A1. ApoE accumulation was blocked by anti-C5 antibody and enhanced by repetitive complement challenge. ApoE mRNA levels were not affected by complement challenge. ApoE was frequently colocalized with MAC in complement-treated cells and drusen from human eyes. ApoE was released into complement-treated conditioned media after a single complement challenge and accumulated on ECM after repetitive complement challenge.Conclusions. Complement challenge induces time-dependent ApoE accumulation in RPE cells. An understanding of the mechanisms by which complement affects RPE ApoE accumulation may help to better explain drusen composition, and provide insights into potential therapeutic targets.

The role of the complement system in cancer.

In addition to being a component of innate immunity and an ancient defense mechanism against invading pathogens, complement activation also participates in the adaptive immune response, inflammation, hemostasis, embryogenesis, and organ repair and development. Activation of the complement system via classical, lectin, or alternative pathways generates anaphylatoxins (C3a and C5a) and membrane attack complex (C5b-9) and opsonizes targeted cells. Complement activation end products and their receptors mediate cell-cell interactions that regulate several biological functions in the extravascular tissue. Signaling of anaphylatoxin receptors or assembly of membrane attack complex promotes cell dedifferentiation, proliferation, and migration in addition to reducing apoptosis. As a result, complement activation in the tumor microenvironment enhances tumor growth and increases metastasis. In this Review, I discuss immune and nonimmune functions of complement proteins and the tumor-promoting effect of complement activation.

Combined inhibition of C5 and CD14 efficiently attenuated the inflammatory response in a porcine model of meningococcal sepsis

BackgroundFulminant meningococcal sepsis, characterized by overwhelming innate immune activation, mostly affects young people and causes high mortality. This study aimed to investigate the effect of targeting two key molecules of innate immunity, complement component C5, and co-receptor CD14 in the Toll-like receptor system, on the inflammatory response in meningococcal sepsis.MethodsMeningococcal sepsis was simulated by continuous intravenous infusion of an escalating dose of heat-inactivated Neisseria meningitidis administered over 3 h. The piglets were randomized, blinded to the investigators, to a positive control group (n = 12) receiving saline and to an interventional group (n = 12) receiving a recombinant anti-CD14 monoclonal antibody together with the C5 inhibitor coversin.ResultsA substantial increase in plasma complement activation in the untreated group was completely abolished in the treatment group (p = 0.006). The following inflammatory mediators were substantially reduced in plasma in the treatment group: Interferon-γ by 75% (p = 0.0001), tumor necrosis factor by 50% (p = 0.01), Interleukin (IL)-8 by 50% (p = 0.03), IL-10 by 40% (p = 0.04), IL-12p40 by 50% (p = 0.03), and granulocyte CD11b (CR3) expression by 20% (p = 0.01).ConclusionInhibition of C5 and CD14 may be beneficial in attenuating the detrimental effects of complement activation and modulating the cytokine storm in patients with fulminant meningococcal sepsis.

Fc microparticles can modulate the physical extent and magnitude of complement activity.

The complement system is an integral component of the humoral immune system, and describes a cascade of interacting proteins responsible for the opsonization and lysis of foreign pathogens, in addition to the recruitment of immune cells. However, complement activation is also implicated in the progression and complication of immune dysfunctions such as sepsis. Microparticle (MP) biomaterials capable of tuning the local magnitude of serum complement activation could improve complement-mediated cytotoxicity to serum-resistant bacteria or calm an overactive immune response during sepsis. We demonstrate that model Fc-functionalized microparticles can be designed to either enhance or diminish the local cytotoxic effect of complement activation in human serum. The particles were formed with either the antibody Fc domains oriented outward from the particle surface or randomly adsorbed in a non-oriented fashion. In the oriented Fc form, complement products were directly sequestered to the particle surface, including C5a, a potent anaphylatoxin that, when elevated, is associated with poor sepsis prognosis. The oriented particle also lowered the cytotoxicity of serum and thus decreased the antibiotic effect when compared to serum alone. Conversely, the non-oriented microparticles were found to sequester similar levels of C5a, but much lower levels of iC3b and TCC on the microparticle surface, thereby increasing the amount of the soluble terminal complement complex. In addition, the non-oriented microparticles extend the distance over which TCC forms and enhance serum cytotoxicity to bacteria. Together, these two types of complement-modulating particles provide the first biomaterial that can functionally modify the range of complement activation at sites distant from the particle surface. Thus, biomaterials that exploit Fc presentation provide new possibilities to functionally modulate complement activation to achieve a desired clinical result.

Terminal complexes of the complement system: new structural insights and their relevance to function

Complement is a key component of innate immunity in health and a powerful driver of inflammation and tissue injury in disease. The biological and pathological effects of complement activation are mediated by activation products. These come in two flavors: (i) proteolytic fragments of complement proteins (C3, C4, C5) generated during activation that bind specific receptors on target cells to mediate effects; (ii) the multimolecular membrane attack complex generated from the five terminal complement proteins that directly binds to and penetrates target cell membranes. Several recent publications have described structural insights that have changed perceptions of the nature of this membrane attack complex. This review will describe these recent advances in understanding of the structure of the membrane attack complex and its by-product the fluid-phase terminal complement complex and relate these new structural insights to functional consequences and cell responses to complement membrane attack.

The role of membrane complement regulatory protein in transplantation of bone marrow mesenchymal stem cell

Objective To explore the effect of complement activation on bone marrow mesenchymal stem cells (BMSCs) and evaluate the effect after transfection of complement regulatory proteins. Methods Bone marrow aspirate was harvested from 10 cases of patients suffered from fractures. Mesenchymal stem cells were isolated, indentified cultured and then experimented in vitro. The complement cytotoxicity on the mesenchymal stem cells in autologous serum was measured by Europium cytotoxicity assay. The samples were divided into BMSCs group, BMSCs+ autologous human serum (AHS) group and BMSCs+ inactivated autologous human serum (iAHS) group. The complement membrane attack complex (MAC) deposited on the membranes was detected by flow cytometry. Finally, the cytotoxicity on BMSCs was measured after transfected with membrane complement regulatory proteins (mCRPs). All samples were divided into BMSCs with mCRPs untransfected group and BMSCs with mCRPs transfected group. Results More than 95% of cells derived from bone marrow were identified to be mesenchymal stem cells through detection of cell surface markers by flow cytometry. The cytotoxicity of untreated cells was 0.41%±1.48%. BMSCs harvested from the 10 patients all had cytotoxicity after incubated with autologous serum, and the cytotoxicity was 32.59%±2.73%, while cytotoxicity after incubated with complement inactivated autologous serum was 2.59%±3.08%, which was similar to control group. Complement attack complex (MAC) could be detected on the BMSCs incubated with autologous serum, which implied the complement activation. After transfection of mCRPs, the cytotoxicity of autologous serum on transfected cells was decreased. The cytotoxicity of untransfected cells (41.70%±4.47%) had significant difference compared to the cells transfected with CD55 (21.87%±2.19%), the cells transfected with CD59 (18.67%±1.42%), and the cells transfected with CD46+CD55+CD59 (28.43%±2.14%). CD55, CD59 and CD46+ CD55+CD59 transfected groups could impair effectively the cytotoxicity from complement. However, the cytotoxicity impairment was less effective in CD46 transfected cells (39.30%±3.96%), which had no significant difference compared to untransfected cells. Conclusion Membrane complement regulatory proteins could effectively protect bone marrow mesenchymal stem cells from attacks by complement. Key words: Bone marrow; Mesenchymal stem cells; Complement membrane attack complex

Abstract A121: Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer

Abstract Background: The complement system is an important arm of the innate immune system, and complement proteins are produced by both cancer cells and cells of the TME. A primary function of the complement system is to remove pathogens and injured cells, and it had been assumed that complement activation would result in elimination of cancer cells. However, recent work has revealed new biologic effects of the complement activation fragments C3a and C5a. For example, C3a and C5a directly stimulate the growth of some tissues. Furthermore, inflammatory cells as well as CD4 and CD8 T cells express receptors for both of these activation fragments (C3aR and C5aR), and a recent study reported that C5a reduces the anti-tumor function of CD8+ T cells. Nevertheless, the role of complement activation in the growth and spread of non-small cell lung cancer is incompletely understood. While complement activation can proceed by multiple pathways, a central event is the deposition of C3 on the cell surface. The goal of this study was to examine the role of complement in lung cancer progression employing an immunocompetent orthotopic model which recapitulates many of the features of human lung cancer. Methods: Two murine lung cancer cell lines derived from C57BL/6 mice were used in an orthotopic model of tumor progression: Lewis Lung Carcinoma (LLC) cells, which are a de-differentiated cell line in which driver mutations have not been identified; and CMT167 cells, which are an epithelial lung cancer cell line that express oncogenic K-Ras. Tumor cells were implanted in the left lungs of syngeneic mice WT C57BL/6 mice, or mice which were globally knocked out for C3 (C3-KO). Primary tumor growth and formation of liver metastases were quantitated in the two groups of mice. Complement activation in the plasma was determined by ELISA. Single cell suspensions of the left lung of tumor-bearing mice were prepared and populations of myeloid cells and immune cells quantitated by flow cytometry. Results: Implantation of cancer lead to systemic complement activation as assessed by ELISA in plasma. Primary tumors and liver metastases were decreased in both cell lines implanted into C3-KO mice compared to controls. However, the degree of inhibition was much greater in CMT167 tumors. LLC cells expressed C3 in vitro, whereas CMT167 cells did not, suggesting that autonomous C3 production by cancer cells may contribute to tumor progression. Recruitment of inflammatory myeloid cells was not altered in tumors growing in C3-KO mice. At early time points, we observed equal increases in T cells (CD4, CD8) in the two groups of mice. However, at later time points (3 weeks), the number of T cells had decreased in WT mice, but remained elevated in C3-KO mice. Conclusions: Complement activation occurs in the setting of tumors. C3 plays a critical role in tumor progression, with strong inhibition observed in a subset of cancer cells. The differential response to complement inhibition may depend on the ability of the cancer cells to produce C3. Effects of complement activation may be mediated by inhibiting cytotoxic T cells. These data suggest that complement inhibitors may represent novel immunotherapeutic targets for lung cancer, and suggest interactions between the complement system and checkpoint inhibitors. Citation Format: Jeff Kwak, Howard Li, Jennifer Laskowski, Mary Weiser-Evans, Joshua Thurman, Raphael A. Nemenoff. Complement activation is critical for tumor progression in an orthotopic immunocompetent model of lung cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A121.