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C3b covalently bound to TeNT diversifies antigenic peptides production. After incubation of B cell either with TeNT or TeNT-C3b, HLA-DR molecules were purified, associated peptides were separated by HPLC. The resulting fractions were collected, lyophilized and incubated with irradiated APC for JASMIN T cell activation as described in Section 2.
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In addition to its well-established role in innate immunity, the complement component C3 is of critical importance in modulating the humoral response. In this study, we examined the effect of C3b linkage to tetanus toxin (TeNT) in the production of antigenic peptides inside human APC. We purified HLA-DR associated peptides isolated either from TeNT...
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Context 1
... h. HLA-DR asso- ciated antigenic peptides were then eluted by HPLC (Fig. 3). The peptides eluted from TeNT and TeNT-C3b fed cells were similar, except for a significant peak at 81 min ( Fig. 3A and B) which was obtained only for TeNT-C3b fed cells. Individual HPLC fractions were then tested for their capacity to stimulate the JASMIN T cell clone (Fig. 4). Whereas a single antigenic peak (72 min) was detected with TeNT pulsed B cell, four major antigenic fractions (70, 75, 81 and 92 min) were obtained from TeNT-C3b pulsed B cells. However, a significant difference in 215 nm profile was observed only at 81 min. Absorbance pro- file corresponds to all peptides present in the mixture: one ...
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Citations
... Self-antigen presentation after degradation of apoptotic cells is guided by activated C3, contributing to MHC class II antigen processing and presentation (72). In several studies, the role of CR1 and 2 in prolonged antigen presentation have been described for antigen-specific B-cells and macrophage-mediated antigen presentation to T-cells (73)(74)(75)(76)(77). Studies have also attempted to describe the mechanistic pathway of antigen-loading efficiency of tetanus toxin-opsonized by C3b and C4b particles (78)(79)(80), though the type of antigen spoken hereof is of pathogenic source and may not necessarily explain mechanisms applicable in the context of trauma. Yet, the dynamics of C3 fragment mediated opsonization in purportedly regulating antigen processing is an intriguing premise. ...
... C3 activation fragments bound to antigen can delay its degradation in the cytosol by evading its fusion with lysosomes and maintain the antigen-loaded internalized cargo within DCs, a process which modulates T-cell responses to self-antigens (72). Additionally, antigens complexed with C3b were shown to form more stable MHC class II complex, modulating the antigen processing pathway in the late endocytic stage, the loading of the antigen onto MHC and the presentation of the antigen to T-cells resulting in T-cell proliferation (72,78,79,81,82). Furthermore, high intracellular cAMP in C3aR-or C3-deficient APCs impaired antigen presentation (83). ...
The overpowering effect of trauma on the immune system is undisputed. Severe trauma is characterized by systemic cytokine generation, activation and dysregulation of systemic inflammatory response complementopathy and coagulopathy, has been immensely instrumental in understanding the underlying mechanisms of the innate immune system during systemic inflammation. The compartmentalized functions of the innate and adaptive immune systems are being gradually recognized as an overlapping, interactive and dynamic system of responsive elements. Nonetheless the current knowledge of the complement cascade and its interaction with adaptive immune response mediators and cells, including T- and B-cells, is limited. In this review, we discuss what is known about the bridging effects of the complement system on the adaptive immune system and which unexplored areas could be crucial in understanding how the complement and adaptive immune systems interact following trauma.
... It is well-established that the formation of covalently linked complexes significantly alters antigen [134][135][136][137][138], producing novel antigenic fragments upon proteolysis and increasing antigenicity. One well-studied case involves complexes between the complement component C3b and tetanus toxin [158,159]. As noted above, it is likely that molecularly complementary complexes of sufficient mutual affinity will behave antigenically in a similar fashion to covalently linked complexes and the case of insulin binding to glucagon was provided as an example. ...
Evidence is reviewed that complementary proteins and peptides form complexes with increased antigencity and/or autoimmunogenicity. Five case studies are highlighted: 1) diphtheria toxin-antitoxin (antibody), which induces immunity to the normally non-antigenic toxin, and autoimmune neuritis; 2) tryptophan peptide of myelin basic protein and muramyl dipeptide ("adjuvant peptide"), which form a complex that induces experimental allergic encephalomyelitis; 3) an insulin and glucagon complex that is far more antigenic than either component individually; 4) various causes of experimental autoimmune myocarditis such as C protein in combination with its antibody, or coxsackie B virus in combination with the coxsackie and adenovirus receptor; 5) influenza A virus haemagglutinin with the outer membrane protein of the Haemophilus influenzae, which increases antigenicity. Several mechanisms cooperate to alter immunogenicity. Complexation alters antigen processing, protecting the components against proteolysis, altering fragmentation and presenting novel antigens to the immune system. Complementary antigens induce complementary adaptive immune responses (complementary antibodies and/or T cell receptors) that produce circulating immune complexes (CIC). CIC stimulate innate immunity. Concurrently, complementary antigens stimulate multiple Toll-like receptors that synergize to over-produce cytokines, which further stimulate adaptive immunity. Thus innate and adaptive immunity form a positive feedback loop. If components of the complex mimic a host protein, then autoimmunity may result. Enhanced antigenicity for production of improved vaccines and/or therapeutic autoimmunity (e.g., against cancer cells) might be achieved by using information from antibody or TCR recognition sites to complement an antigen; by panning for complements in randomized peptide libraries; or using antisense peptide strategies to design complements.
... Plasma proteins play a critical role in mediating inflammation and innate immunity. Circulating levels of complement, defense collagens, and cytokines are essential in the immune response, playing roles such as modulation of antigen presentation and T/B-cell maturation/differentiation [21][22][23][24]. Moreover, soluble innate immune molecules have emerged as important molecules in HIV and HCV pathogenesis [25,26]. ...
Background:
HIV infection contributes to accelerated rates of progression of liver fibrosis during hepatitis C virus (HCV) infection, and HCV liver disease contributes to mortality during HIV infection. Although mechanisms underlying these interactions are not well known, soluble and cellular markers of immune activation associate with disease progression during both infections.
Methods:
We identified proteins varying in expression across the plasma proteomes of subjects with untreated HIV infection, untreated HCV infection with low aspartate transaminase/platelet ratio index, untreated HCV infection with high aspartate transaminase/platelet ratio index, HIV-HCV coinfection, and controls. We examined correlations between dysregulated proteins and markers of immune activation to uncover biomarkers specific to disease states.
Results:
We observed the anticipated higher frequencies of HLA-DRCD38CD4 and CD8 T cells, higher serum soluble CD14 levels, and higher serum interleukin-6 levels for HCV- and HIV-infected groups compared with controls. Plasma proteome analysis identified 2297 peptides mapping to 227 proteins, and quantitative analysis of peptide intensity identified significant changes in 85 proteins across the 5 groups. Abundance for 7 of these proteins was validated by enzyme-linked immunosorbent assay. Forty-three of these proteins correlated with markers of immune activation, including at least 2 proteins that may directly drive T-cell activation. As a functional validation, we tested the enzymatic pathway product (lysophosphatidic acid, LPA) of one such protein, ecotonucleotide pyrophosphatase/phosphodiesterase-2, for ability to activate T cells in vitro. LPA activated T cells to express CD38 and HLA-DR.
Conclusions:
These data indicate that elevated levels of ecotonucleotide pyrophosphatase/phosphodiesterase-2 and LPA during advanced HCV disease may play a role in exacerbating immune activation during HCV-HIV coinfection.
... The innate immune system is highly integrated with adaptive immunity. Soluble innate molecules can modulate antigen presentation353637, directing the specificity of T cells and antibodies. In turn, antibodies can trigger and modulate innate antiviral effector mechanisms, contributing to enhanced antigen presentation [38]. ...
Infection with Hepatitis C Virus (HCV) causes chronic disease in approximately 80% of cases, resulting in chronic inflammation and cirrhosis. Current treatments are not completely effective, and a vaccine has yet to be developed. Spontaneous resolution of infection is associated with effective host adaptive immunity to HCV, including production of both HCV-specific T cells and neutralizing antibodies. However, the supporting role of soluble innate factors in protection against HCV is less well understood. The innate immune system provides an immediate line of defense against infections, triggering inflammation and playing a critical role in activating adaptive immunity. Innate immunity comprises both cellular and humoral components, the humoral arm consisting of pattern recognition molecules such as complement C1q, collectins and ficolins. These molecules activate the complement cascade, neutralize pathogens, and recruit antigen presenting cells. Here we review the current understanding of anti-viral components of the humoral innate immune system that play a similar role to antibodies, describing their role in immunity to HCV and their potential contribution to HCV pathogenesis.
... When activated by limited proteolysis the thioester becomes extremely reactive with a very short serum lifetime, and will react rapidly with many nucleophiles (Sim and Law 1985;). Conjugation of C3b to ovalbumin and other targets (Cretin et al. 2007; Villiers et al. 1999) has been achieved by adding trypsin to C3 in an excess of the conjugating agent to take advantage of this activation, although coupling efficiency is low. Radiolabelled methylamine has been incorporated into serum fractions of many organisms to screen for the presence of thioester proteins across metazoans (). ...
... With the demonstration of efficient ligation of labels to the thioester proteins other potential applications can be envisaged. C3 (as C3b and C3d) is an excellent natural, non-toxic adjuvant (Cretin et al. 2007; Villiers et al. 1999 ). Many efforts have been made to selectively incorporate antigen to C3 or its processed derivatives. ...
The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum.
... Correspondingly, C3b opsonization augments protein antigen uptake [65,66] and T cell stimulation [65,67,68]. Covalent C3b modification can target antigen to specific MHC class II containing vesicles [69] and may increase lysosomal peptide-MHC stability [70], and the diversity of T cell epitopes presented [71]. Additionally, a deficiency of C1q can lead to suboptimal antigen uptake, impaired DC differentiation and maturation, and reduced T cell responses [64,[72][73][74][75][76][77]. ...
The complement system is a family of serum and cell surface proteins that recognize pathogen-associated molecular patterns, altered-self ligands, and immune complexes. Activation of the complement cascade triggers several antiviral functions including pathogen opsonization and/or lysis, and priming of adaptive immune responses. In this review, we will examine the role of complement activation in protection and/or pathogenesis against infection by Flaviviruses, with an emphasis on experiments with West Nile and Dengue viruses.
... Previous studies have illustrated the role of C3 in Ag presentation (Jacquier-Sarlin et al., 1995;Rey-Millet et al., 1994;Santoro et al., 1994), and particularly in Ag degradation: indeed, JacquierSarlin et al. reported limited TT proteolysis by B cells in the presence of C3b (Jacquier Sarlin et al., 1995). Furthermore, antigenic peptides production and loading on MHC II molecules are modified upon covalent binding of C3b to the Ag ( Cretin et al., 2007;Serra et al., 1997). It has been shown that the TT-C3b interaction is fairly stable in endosomes, whereas it is gradually disrupted in lysosomes (Rey-Millet et al., 1994). ...
... In conclusion, our results, by revealing a novel role for C3, supplement the integrative view on the modulatory role of C3 in acquired immune response, and especially on Ag processing: C3 acts at different levels such as Ag uptake (Villiers et al., 1996), intracellular Ag trafficking and targeting ), proteolytic machinery of Ag processing (these results), Ag peptide editing ( Cretin et al., 2007) and loading on MHC II molecules ( Serra et al., 1997), all of these increasing Ag presentation efficiency (Jacquier-Sarlin et al., 1995) and thus the quality of the acquired immune response (Dempsey et al., 1996;Villiers et al., 1999). 125 I-TT (5 µg) was incubated for 18 h at 37°C in the presence or absence of LF from U937 cells (2.5 x 10 6 cells) and of C3 (5 µg). ...
Increasing evidence underlines the involvement of complement component C3 in the establishment of acquired immunity which appears to play a complex role and to act at different levels. As antigen proteolysis by antigen presenting cells is a key event in the control of antigen presentation efficiency, and consequently in the quality of the immune response, we investigated whether C3 could modulate this step. Our results demonstrate for the first time that C3 can interfere with antigen proteolysis: (i) proteolysis of tetanus toxin (TT) by the lysosomal fraction from a human monocytic cell line (U937) is impaired in the presence of C3, (ii) this effect is C3-specific and involves the C3c fragment of the protein, (iii) C3c is effective even after disulfide disruption, but none of its three constitutive peptides is individually accountable for this inhibitory effect and (iv) the target-protease(s) exhibit(s) a serine-protease activity. The physiological relevance of our results is demonstrated by experiments showing a subcellular colocalisation of TT and C3 after their uptake by U937 and the reduction of TT proteolysis once internalised together with C3. These results highlight a novel role for C3 that broadens its capacity to modulate acquired immune response.
The complement system is a family of more than 30 serum proteins and cell surface receptors that recognizes pathogen-associated
molecular patterns, altered-self ligands, and immune complexes. Complement activation through the classical, lectin and alternative
pathways triggers an array of functions including direct pathogen opsonization and/or lysis, and enhancement of B and T cell
responses. Recent work has demonstrated that complement activation is necessary for the development of protective immune responses
and mediates important antiviral effector functions of Flavivirus-specific antibody. In this review, we will examine the mechanisms
and functions of complement activation and discuss our current understanding of the role of complement in protection against
infection by Flaviviruses, with an emphasis on experiments with West Nile virus (WNV). In addition, we will discuss ongoing
studies on the immune evasion mechanisms that WNV uses to limit complement activation.
KeywordsComplement-immunology flaviviruses-pathogenesis
Complement C3 is a central component of the humoral immune system. Upon triggering of the complement cascade, proteolytic fragments of C3 mediate important processes such as opsonization and lymphocyte activation. C3 possesses an internal thioester that mediates covalent attachment of proteolytically activated C3 to target surfaces. Treatment of native C3 with methylamine cleaves the thioester bond and exposes a free sulfhydryl group at the target-binding face of the protein. Through the use of sulfhydryl-reactive heterobifunctional cross-linking and biotinylation reagents, we demonstrate the capacity to form stable, multimeric whole human C3-protein conjugates in a fashion reflecting the orientation of physiologically-activated C3. We speculate that this C3 conjugation strategy presents a route for targeting dendritic cells and macrophages. In addition, manipulation of the thioester bond could enhance the study of biological roles of C3 and related proteins such as C4, and also of transmissible agents that exploit complement function such as prions.
