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C3 is a key complement protein, located at the nexus of all complement activation pathways. Extracellular, tissue, cell-derived, and intracellular C3 plays critical roles in the immune response that is dysregulated in many diseases, making it an attractive therapeutic target. However, challenges such as very high concentration in blood, increased acute expression, and the elevated risk of infections have historically posed significant challenges in the development of C3-targeted therapeutics. This is further complicated because C3 activation fragments and their receptors trigger a complex network of downstream effects; therefore, a clear understanding of these is needed to provide context for a better understanding of the mechanism of action (MoA) of C3 inhibitors, such as pegcetacoplan. Because of C3's differential upstream position to C5 in the complement cascade, there are mechanistic differences between pegcetacoplan and eculizumab that determine their efficacy in patients with paroxysmal nocturnal hemoglobinuria. In this review, we compare the MoA of pegcetacoplan and eculizumab in paroxysmal nocturnal hemoglobinuria and discuss the complement-mediated disease that might be amenable to C3 inhibition. We further discuss the current state and outlook for C3-targeted therapeutics and provide our perspective on which diseases might be the next success stories in the C3 therapeutics journey.

While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the ‘complosome’, functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also non-redundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal-sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux towards reactive oxygen species (ROS) generation and anaerobic glycolysis to favor IL-1β production, both at the transcriptional level and processing of pro-IL-1β. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

Complement was initially discovered as an assembly of plasma proteins “complementing” the cytolytic activity of antibodies. However, our current knowledge places this complex system of several plasma proteins, receptors, and regulators in the center of innate immunity as a bridge between the initial innate responses and adaptive immune reactions. Consequently, complement appears to be pivotal for elimination of pathogens, not only as an early response defense, but by directing the subsequent adaptive immune response. The discovery of functional intracellular complement and its roles in cellular metabolism opened novel avenues for research and potential therapeutic implications. The recent studies demonstrating immunoregulatory functions of complement in the tumor microenvironment and the premetastatic niche shifted the paradigm on our understanding of functions of the complement system in regulating immunity. Several complement proteins, through their interaction with cells in the tumor microenvironment and in metastasis-targeted organs, contribute to modulating tumor growth, antitumor immunity, angiogenesis, and therefore, the overall progression of malignancy and, perhaps, responsiveness of cancer to different therapies. Here, we focus on recent progress in our understanding of immunostimulatory vs. immunoregulatory functions of complement and potential applications of these findings to the design of novel therapies for cancer patients.

The complement system is an evolutionarily ancient key component of innate immunity required for the detection and removal of invading pathogens. It was discovered more than 100 years ago and was originally defined as a liver-derived, blood-circulating sentinel system that classically mediates the opsonization and lytic killing of dangerous microbes and the initiation of the general inflammatory reaction. More recently, complement has also emerged as a critical player in adaptive immunity via its ability to instruct both B and T cell responses. In particular, work on the impact of complement on T cell responses led to the surprising discoveries that the complement system also functions within cells and is involved in regulating basic cellular processes, predominantly those of metabolic nature. Here, we review current knowledge about complement's role in T cell biology, with a focus on the novel intracellular and noncanonical activities of this ancient system.

Recent workdefined an unexpected and critical role for intracellular/autocrine active complement in human Th1 biology. Specifically, autocrine stimulation of CD46via TCR-induced C3b generation is needed for IFN-γ production by human CD4+ T cells. Thus, CD46-deficient patients are unable to generate normal Th1 responses. Analysis of the CD46-driven gene signature in Th1 cells revealed CD109 as a direct target of CD46. Indeed, T cells from CD46 deficient patients and from healthy donors in which CD46 expression was ablated via CRISPR-Cas9 technology failed to upregulate CD109. CD109 is aGPI-linked surface protein, mostly expressed on non-immune cells, and belongs to the complement C3 protein family. Though CD109 has been reported as a major negative regulator of TGF-β receptor signaling, direct functional activity for of CD109 on T cells remains unexplored. Knock-out of CD109 in human CD4+ T cells with CRISPR-Cas9 technology induced uncontrolled Th1 and Th17 activation upon in vitro stimulation under non-skewing conditions. Similarly, CD4+T cells from Cd109−/− mice displayed augmented Th1 and Th17 in vitro responses and caused significantly more tissue pathology and weight loss in a T cell-transfer colitis model. Surprisingly, on T cells, CD109 does not restrain IFN-γ and/or IL-17 induction via modulation of TGF-β receptor activity. Instead, CD109 engages with a non-canonical costimulatory molecule and controls stemness- and metabolism-related signaling pathways. Together, these data suggest that the complement-related protein CD109 serves as an important and novel molecular switch on CD4+ T-cells, where it regulates the balance between Th1 and Th17-induction pathways.

Highlights • Recent, evidence have demonstrated presence of intracellular complement proteins and their activation (the complosome). • The complosome plays a role in basic cell processes such as metabolism. • The interaction between the complosome and intracellular pathogens needs to be further elucidated., The complement system was defined over a century ago based on its ability to “complement” the antibody-mediated and cell-mediated immune responses against pathogens. Today our understanding of this ancient part of innate immunity has changed substantially and we know now that complement plays an undisputed pivotal role in the regulation of both innate and adaptive immunity. The complement system consists of over 50 blood-circulating, cell-surface expressed and intracellular proteins. It is key in the recognition and elimination of invading pathogens, also in the removal of self-derived danger such as apoptotic cells, and it supports innate immune responses and the initiation of the general inflammatory reactions. The long prevailing classic view of complement was that of a serum-operative danger sensor and first line of defence system, however, recent experimental and clinical evidences have demonstrated that “local” tissue and surprisingly intracellular complement (the complosome) activation impacts on normal cell physiology. This review will focus on novel aspects of intracellular complement activation and its unexpected roles in basic cell processes such as metabolism. We also discuss what the existence of the complosome potentially means for how the host handles intracellular pathogens such as viruses.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Anti-neutrophil cytoplasmic antibody vasculitis is a systemic autoimmune disease with glomerulonephritis and pulmonary haemorrhage as major clinical manifestations. The name reflects the presence of autoantibodies to myeloperoxidase and proteinase-3, which bind to both neutrophils and monocytes. Evidence of the pathogenicity of these autoantibodies is provided by the observation that injection of anti-myeloperoxidase antibodies into mice causes a pauci-immune focal segmental necrotizing glomerulonephritis which is histologically similar to the changes seen on renal biopsy in patients. Previous studies in this model have implicated the alternative pathway of complement activation and the anaphylatoxin C5a. Despite this progress, the factors that initiate complement activation have not been defined. In addition, the relative importance of bone marrow-derived and circulating C5 is not known. This is of interest given the recently identified roles for complement within leukocytes. We induced anti-myeloperoxidase vasculitis in mice and confirmed a role for complement activation by demonstrating protection in C3-deficient mice. We showed that neither MASP-2- nor properdin-deficient mice were protected, suggesting that alternative pathway activation does not require properdin or the lectin pathway. We induced disease in bone marrow chimaeric mice and found that circulating and not bone marrow-derived C5 was required for disease. We have therefore excluded properdin and the lectin pathway as initiators of complement activation and this means that future work should be directed at other potential factors within diseased tissue. In addition, in view of our finding that circulating and not bone marrow-derived C5 mediates disease, therapies that decrease hepatic C5 secretion may be considered as an alternative to those that target C5 and C5a. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Intracellularly generated and autocrine-functioning complement component C3 is a critical integral part of normal human CD4+ Th1 and cytotoxic CD8+ T cells responses. Increased or decreased intracellular C3 result in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression are, however, undefined. By comparing transcriptomes from blood and tissue, we identified the complement system, including C3, as one of the most significantly enriched biological pathways in tissue-occupying cells of human macrophages, CD4+ and CD8+ T cells. By leveraging a novel C3 reporter mouse, we confirmed that C3 gene transcription is a feature of immune cells in tissues, is induced during trans-endothelial diapedesis, and is dependent on the integrin intercellular adhesion molecule (ICAM)-1 liganding lymphocyte function-associated antigen (LFA)-1. Consequently, monocytes and T cells from patients with leukocyte adhesion deficiency (LAD)-1 had reduced C3 and diminished effector activities, which could be rescued proportionally by normalization of intracellular C3. In synovia of patients with rheumatoid arthritis (RA), C3 transcript expression by CD4+ T cells was linearly associated with disease severity and acted as a biomarker distinguishing inflamed versus uninflamed RA. Our study defines the integrin network as a novel and key controller of intracellular complement, demonstrates that perturbations in the LFA-1–C3 axis contribute to primary human immunodeficiency and identifies T cell C3 production as a biomarker of the severity of autoimmunity.

Intracellular/autocrine complement proteins have emerged as critical regulators of human Th1 induction and contraction. T cells contain both intracellular C3 and C5 activation systems, with intracellular C3aR1 and C5aR1 stimulation driving T cell homeostatic survival and normal Th1 IFN-gamma production, respectively. Here we demonstrate how the intracellular/autocrine C5 system is regulated by using T cells from the first described patient with C5aR2 deficiency. This patient suffers from an autoinflammatory syndrome, with enhanced inflammatory Th responses and a profound loss of naïve CD4 T cells (95% of CD4 T cells in the blood have a memory T cell phenotype). Thus, in contrast to intracellular C5aR1 stimulation, cell surface expressed C5aR2 is an important negative regulator of inflammatory Th induction. While both C5aR1 and C5aR2 can bind C5a, we found that the carboxypeptidase-processed form of C5a, C5a-desArg, was twice as potent as C5a in reducing Th1 induction. In addition, carboxypeptidase M (CPM) expression was highly induced upon T cell activation indicating that CPM may be mediating T cell-derived C5a-desArg generation and thus, C5aR2 stimulation. In this vein, activation of T cells in the presence of a CPM inhibitor induced Th1 hyper-induction, which was rescued by addition of a C5aR2 agonist and reduced by adding C5a-desArg, but not C5a. The in vivo importance of T cell-expressed CPM and autocrine C5aR2 activation is demonstrated by the fact that CPM−/− mice have enhanced inflammatory Th responses and CPM−/− T cells cause increased pathology in an in vivo T cell transfer colitis model. These data highlight the complex auto-regulatory functions of complement in T cell responses.

For a relatively long period of time, mental functioning was mainly associated with personal profile while brain functioning went by the wayside. After the 90s of the 20th century, or the so called "Decade of the Brain", today, contemporary specialists work on the boundary between fundamental science and medicine. This brings neuroscience, neuropsychology, psychiatry, and psychotherapy closer to each other. Today, we definitely know that brain structures are being built and altered thanks to experience. Psychotherapy can be more effective when based on a neuropsychological approach-this implies identification of the neural foundations of various disorders and will lead to specific psychotherapeutic conclusions. The knowledge about the brain is continually enriched, which leads to periodic rethinking and updating of the therapeutic approaches to various diseases of the nervous system and brain dysfunctions. The aim of translational studies is to match and combine scientific areas, resources, experience and techniques to improve prevention, diagnosis and therapies, and "transformation" of scientific discoveries into potential treatments of various diseases done in laboratory conditions. Neuropsychological studies prove that cognition is a key element that links together brain functioning and behaviour. According to Dr. Kandel, all experimental events, including psychotherapeutic interventions, affect the structure and function of neuronal synapses. The story of why psychotherapy works is a story of understanding the brain mechanisms of psychic processes, a story of how the brain has been evolving to ensure learning, forgetting, and the mechanisms of permanent psychological change. The new evidence on brain functioning necessitates the integration of neuropsychological achievements in the psychotherapeutic process. An integrative approach is needed to take into account the dynamic interaction between brain functioning, psyche, soul, spirit, and social interaction, ie, development of a model of psychotherapeutic work based on cerebral plasticity! Brain-based psychotherapy aims at changing brain functioning not directly, but through experiences. This is neuro-psychologically informed psychotherapy.

The complement component C3 is the major effector molecule of the complement system. C3 circulates in the blood and interstitial fluids as pro-enzyme and is activated by enzymatic cleavage into a C3a portion, a classic anaphylatoxin that functions as chemoattractant and immune cell activator, and the C3b portion, the body's most potent opsonin. C3 cleavage is in most cases mediated by an enzyme complex called the C3 convertase. However, it is now becoming increasingly clear that the cleavage of C3 by a range of 'single' proteases into bioactive C3a and C3b fragments is of high physiological significance. Here, we describe a protocol for the enzymatic cleavage of human C3 by the serine protease cathepsin L and the detection of the cleavage products C3a and C3b by western blotting as an example for this kind of enzymatic reactions.

Complement is traditionally known to be a system of serum proteins that provide protection against pathogens through direct cell lysis and the mobilization of innate and adaptive immunity. However, recent work indicates that the complement system has additional physiological roles beyond those in host defence. In this Opinion article, we describe the new modes and locations of complement activation that enable it to interact with other cell effector systems, such as growth factor receptors, inflammasomes and metabolic pathways. We propose that the location of complement activation dictates its function.

Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance., Graphical Abstract, Highlights • Complement C3 is activated intracellularly in human T cells by cathepsin L • Intracellular C3 activation mediates cell survival and Th1 induction • Increased intracellular C3 activation underlies T effector dysregulation in arthritis • Patients with serum C3-deficiency retain intracellular C3a generation

Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Recently, we designed a peptide derived from the neural-restrictive silencer factor (REST), REST68, which we showed to inhibit expression of CD59 in tumors lacking the full-length REST and proposed a detailed model for regulation of CD59 expression via interplay between REST and nucleolin (NCL) transcription factors. In this paper, we study in detail the mechanisms for sensitization of malignant cells to Ab-based cancer immunotherapy by the REST68 peptide and the implications of the REST/NCL model for the design of treatment resulting in higher tumor susceptibility. REST68 inhibited CD59 expression in malignant cells expressing either truncated or full-length REST, but not in nonmalignant cells. However, activation of protein kinase C (PKC) in nonmalignant cells, a process that contributes to cellular transformation, phosphorylated NCL and enabled suppression of CD59 expression by the REST68. Combined treatment of different tumor types with REST68 and PKC inhibitor synergized to further suppress CD59 expression and reduce resistance to complement lysis. The combined treatment also increased susceptibility of tumors expressing either of the REST isoforms to PBMC-mediated killing, which, at least in part, accounted for the strong promotion of apoptosis by the REST68/PKC inhibitor. These data demonstrate that REST68 sensitizes tumors to Ab-based cancer immunotherapy via multiple mechanisms. Furthermore, the REST/NCL interplay model for regulation of expression of cd59 and other genes involved in cell survival enables the design of treatments for different tumor types to achieve more efficient tumor clearance.

Alzheimer’s disease (AD) is an age-related neurodegenerative disease that affects approximately 24 million people worldwide. A number\ud of different risk factors have been implicated in AD; however, neuritic (amyloid) plaques are considered as one of the defining risk\ud factors and pathological hallmarks of the disease. In the past decade, enormous efforts have been devoted to understand the genetics\ud and molecular pathogenesis leading to neuronal death in AD, which has been transferred into extensive experimental approaches aimed\ud at reversing disease progression. Modern medicine is facing an increasing number of treatments available for vascular and neurodegenerative\ud brain diseases, but no causal or neuroprotective treatment has yet been established. Almost all neurological conditions are characterized\ud by progressive neuronal dysfunction, which, regardless of the pathogenetic mechanism, finally leads to neuronal death. The\ud particular emphasis of this review is on risk factors and mechanisms resulting in neuronal loss in AD and current and prospective opportunities\ud for therapeutic interventions. This review discusses these issues with a view to inspiring the development of new agents that\ud could be useful for the treatment of AD.

Udgivelsesdato: 2009-Jun Mannan-binding lectin (MBL) is a plasma protein implicated in innate immune defence against a broad range of microorganisms, including viruses. It is also thought that MBL plays a role in the recruitment of the specific clonal immune response. This was studied by injecting soluble hepatitis B surface antigen (HBsAg) intravenously into mice deficient in both MBL-A and MBL-C (MBL DKO mice). The MBL DKO animals on mixed genetic background (SV129EvSv x C57BL/6) produced higher antibody titres than the wild-type littermates. After primary challenge with the antigen the immunoglobulin M anti-HBsAg antibody titres were threefold higher in the MBL DKO mice than in the wild-type mice. Following the boost, the immunoglobulin G anti-HBsAg antibody titres were 10-fold higher in the MBL DKO mice, suggesting that MBL plays a role in a negative feedback regulation of adaptive immunity. However, the modulating effect of MBL was dependent on the genetic environment. The MBL DKO mice backcrossed on a C57BL/6 background showed the opposite response with the MBL DKO mice now producing fewer antibodies than the wild-type animals, whereas MBL deficiency in mice with the SV129EvSv background did not show any effect in antibody production. These findings indicate that the modifying effect of MBL on the humoral immune response is influenced by the genetic environment.

Mannan-binding lectin-associated serine protease 2 (MASP-2) is an enzyme of the innate immune system. MASP-2 forms complexes with the pattern recognition molecules mannan-binding lectin (MBL), H-ficolin, L-ficolin, or M-ficolin, and is activated when one of these proteins recognizes microorganisms and subsequently cleaves complement factors C4 and C2, thus initiating the activation of the complement system. Missense polymorphisms of MASP-2 exist in different ethnic populations. To further characterize the nature of these, we have produced and characterized rMASP-2s representing the following naturally occurring polymorphisms: R99Q, D120G, P126L, H155R, 156_159dupCHNH (CHNHdup), V377A, and R439H. Only very low levels of CHNHdup were secreted from the cells, whereas quantities similar to wild-type MASP-2 were found intracellularly, indicating that this mutation results in a misfolded protein. We found that D120G and CHNHdup could not associate with MBL, whereas R99Q, P126L, H155R, V377A, R439H, and wild-type MASP-2 bound equally well to MBL. Accordingly, when D120G and CHNHdup were mixed with MBL, no activation of complement factor C4 was observed, whereas R99Q, P126L, and V377A cleaved C4 with an activity comparable to wild-type MASP-2 and H155R slightly better. In contrast, the R439H variant was deficient in this process despite its normal binding to MBL. This variant was also not able to autoactivate in the presence of MBL and mannan. We find the R439H variant is common in Sub-Saharan Africans with a gene frequency of 10%. Our results indicate that individuals with different types of MASP-2 defects may be identified through genotyping.

C1q is the first subcomponent of the classical complement pathway that binds antigen-bound IgG or IgM and initiates complement activation via association of serine proteases C1r and C1s. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has some structural and functional autonomy. Although a number of studies have tried to identify IgG-binding sites on the gC1q domain, no such attempt has been made to localize IgM-binding site. On the basis of the information available via the gC1q crystal structure, molecular modeling, mutational studies, and bioinformatics, we have generated a series of substitution mutants of ghA, ghB, and ghC and examined their interactions with IgM. The comparative analysis of IgM- and IgG-binding abilities of the mutants suggests that the IgG- and IgM-binding sites within the gC1q domain are different but may overlap. Whereas Arg(B108), Arg (B109), and Tyr(B175) mainly constitute the IgM-binding site, the residues Arg(B114), Arg(B129), Arg(B163), and His(B117) that have been shown to be central to IgG binding are not important for the C1q-IgM interaction. Given the location of Arg(B108), Arg (B109), and Tyr(B175) in the gC1q crystal structure, it is likely that C1q interacts with IgM via the top of the gC1q domain.

C1q is the first subcomponent of the classical complement pathway that can interact with a range of biochemically and structurally diverse self and nonself ligands. The globular domain of C1q (gC1q), which is the ligand-recognition domain, is a heterotrimeric structure composed of the C-terminal regions of A (ghA), B (ghB), and C (ghC) chains. The expression and functional characterization of ghA, ghB, and ghC modules have revealed that each chain has specific and differential binding properties toward C1q ligands. It is largely considered that C1q-ligand interactions are ionic in nature; however, the complementary ligand-binding sites on C1q and the mechanisms of interactions are still unclear. To identify the residues on the gC1q domain that are likely to be involved in ligand recognition, we have generated a number of substitution mutants of ghA, ghB, and ghC modules and examined their interactions with three selected ligands: IgG1, C-reactive protein (CRP), and pentraxin 3 (PTX3). Our results suggest that charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side of the ghB are crucial for C1q binding to these ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. Thus, a recently proposed model suggesting the rotation of the gC1q domain upon ligand recognition may be extended to C1q interaction with CRP and PTX3 in addition to IgG1.

SummaryExpansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. Here we found that in activated human T cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T cells from CD46-deficient patients, characterized by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN-γ production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4+ T cell effector function.

CD46 is an important regulator of the complement system by preventing unwanted deposition of the complement activation products and opsonins C3b/C4b onto self-tissue. Recently, intracellular signals mediated by CD46 activation on several distinct human cell types have demonstrated that CD46 also plays decisive roles in immuneregulation. The growing recognition of CD46 as key regulator in several vital biological processes, led to increased demand in sensitive methods for monitoring CD46 expression and changes thereof on cells and in tissues. Here we describe a method, which allows for studying CD46 expression on the surface of cells using specific antibodies in combination with fluorescence-activated cell sorting (FACS) analysis.

CD59 is the single regulator of the terminal complement pathway. It has been implicated in disease such as Paroxysmal nocturnal hemoglobinuria (PMH) and cancer. Expression of CD59 protects normal and malignant cells from the cytotoxic potential of the complement system. Here we describe a method, which allows for studying its expression on the surface of cells.

CD59 overexpression has been shown to confer the resistance of tumors to complement lysis. Complement lysis is one of the two major killing mechanisms of therapeutic anticancer antibodies. This chapter provides a method that allows studying the extent of complement protection of tumors by CD59.

CD46 is an important regulator of the complement system by preventing unwanted deposition of the complement activation products and opsonins C3b/C4b onto self-tissue. Recently, intracellular signals mediated by CD46 activation on several distinct human cell types have demonstrated that CD46 also plays decisive roles in immuneregulation. The growing recognition of CD46 as key regulator in several vital biological processes, led to increased demand in sensitive methods for monitoring CD46 expression and changes thereof on cells and in tissues. Here we describe a method, which allows for studying CD46 expression on the surface of cells using specific antibodies in combination with fluorescence-activated cell sorting (FACS) analysis.

CD59 is the single regulator of the terminal complement pathway. It has been implicated in disease such as Paroxysmal nocturnal hemoglobinuria (PMH) and cancer. Expression of CD59 protects normal and malignant cells from the cytotoxic potential of the complement system. Here we describe a method, which allows for studying its expression on the surface of cells.

Review on CD59 (CD59 molecule, complement regulatory protein), with data on DNA, on the protein encoded, and where the gene is implicated.

The complement system is among the evolutionary oldest ‘players’ of the immune system. It was discovered in 1896 by Jules Bordet as a heat-labile fraction of the serum responsible for the opsonisation and subsequent killing of bacteria. The decades between the 1920s and 1990s then marked the discovery and biochemical characterization of the proteins comprising the complement system. Today, complement is defined as a complex system consisting of more than 30 membrane-bound and soluble plasma proteins, which are activated in a cascade-like manner, very similarly to the caspase proteases and blood coagulation systems. Complement is engrained in the immunologist's mind as a serum-effective, quintessential part of innate immunity, vitally required for the detection and removal of pathogens or other dangerous entities. Three decades ago, this rather confined definition was challenged and then refined when it was shown that complement participates vitally in the induction and regulation of B cell responses, thus adaptive immunity. Similarly, research work published in more recent years supports an equally important role for the complement system in shaping T cell responses. Today, we are again facing paradigm shifts in the field: complement is actively involved in the negative control of T cell effector immune responses, and thus, by definition in immune homeostasis. Further, while serum complement activity is without doubt fundamental in the defence against invading pathogens, local immune cell-derived production of complement emerges as key mediator of complement's impact on adaptive immune responses. And finally, the impact of complement on metabolic pathways and the crosstalk between complement and other immune effector systems is likely more extensive than previously anticipated and is fertile ground for future discoveries. In this review, we will discuss these emerging new roles of complement, with a focus on Th1 cell biology.

Recently, there has been an increase of interest in the use of biological or immune-based therapies for patients with malignancies. This has been informed by the deeper understanding of the crosstalk between the host immune system and malignant tumours, as well as the potential advantages of immunotherapy—high specificity and less toxicity compared to standard approaches. The particular emphasis of this article is on the role of the complement system in tumour growth and antibody-based cancer immunotherapy. The functional consequences from overexpression of complement regulators by tumours and the development of strategies for overcoming this are discussed in detail. This review discusses these issues with a view to inspiring the development of new agents that could be useful for the treatment of cancer.

An increasing number of studies have shown a critical role for the membrane attack complex, synthesized on activation of the terminal pathway of the complement system, in causing demyelination and neuronal death in neurodegeneration. The aim of this study was to develop a strategy to increase the resistance of neurons to complement damage by modulating the expression of membrane complement regulatory protein CD59, the only inhibitor of the terminal pathway of the complement cascade. We exploited our recent finding that CD59 expression is regulated by the neural-restrictive silencer factor (REST) and designed a novel REST-derived peptide (REST5) containing the nuclear localization domain of the wild-type protein. The effect of REST5 and the mechanism by which it modulates CD59 expression were modelled in neuroblastoma cells transfected with expression constructs, and then confirmed in human neurons differentiated from neural progenitor cells. REST5 increased the expression of CD59 in neurons by fivefold and protected them from complement-mediated lysis spontaneously triggered by neurons. As a source of complement, we used either human serum or conditioned medium from primary human oligodendroglia. This study brings new insight into immunopharmacological research that may serve to inhibit neuronal death triggered by the terminal pathway of complement activation.

Alzheimer's disease (AD) is an age-related neurodegenerative disease that affects approximately 24 million people worldwide. A number of different risk factors have been implicated in AD, however, neuritic (amyloid) plaques are considered as one of the defining risk factors and pathological hallmarks of the disease. Complement proteins are integral components of amyloid plaques and cerebral vascular amyloid in Alzheimer brains. They can be found at the earliest stages of amyloid deposition and their activation coincides with the clinical expression of Alzheimer's dementia. This review emphasizes on the dual key roles of complement system and complement regulators (CRegs) in disease pathology and progression. The particular focus of this review is on currently evolving strategies for design of complement inhibitors that might aid therapy by restoring the fine balance between activated components of complement system, thus improving the cognitive performance of patients. This review discusses these issues with a view to inspiring the development of new agents that could be useful for the treatment of AD.

Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases.