The paradoxical roles of C1q and C3 in autoimmunity

Diane Scott and Marina Botto

Centre for Complement and Inflammation Research, Department of Medicine,

Imperial College London, London, UK

Corresponding Author

Prof Marina Botto

Centre for Complement and Inflammation Research,

Department of Medicine, Imperial College London

Du Cane Road, London W12 0NN, UK

Tel.: +44-(0)20 3313 2316

Fax: +44-(0)20 3313 2379

E-mail: m.botto@imperial.ac.uk

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In this review we will focus on the links between complement and

autoimmune diseases and will highlight how animal models have provided insights

into the manner by which C1q and C3 act to modulate both adaptive and innate

immune responses. In particular we will highlight how C1q may not only act as

initiator of the classical complement pathway, but can also mediate multiple immune

responses in a complement activation independent manner.

The paradoxical role of complement in autoimmunity

Complement has been shown to contribute to the immunopathology of several

autoimmune diseases including systemic lupus erythematosus (SLE), anti glomerular

basement membrane (anti-GBM) disease and rheumatoid arthritis (RA) (Ballanti et

al., 2013; Chen et al., 2010). Notwithstanding that, and somewhat paradoxically,

complement also appears able to protect against autoimmunity since complement

deficiencies, particularly of the early components of the classical pathway, are

associated with autoimmune disorders, particularly SLE (Pickering et al., 2000)

SLE is a systemic autoimmune disease characterised by circulating

autoantibodies and low complement levels. A proposed model to explain the immune

pathology is that autoantigen/autoantibody complexes become deposited at various

affected sites where they cause complement activation and organ damage. It might

therefore be expected that complement deficiencies would protect against this

condition. However, this is not the case and indeed studies of rare homozygous

complement deficiencies have shown that the early components of the classical

pathway are important in protection against this disease. These studies showed a very

strong concordance (>90%) of SLE between siblings with C1q, C1r/C1s and C4

deficiencies (Pickering et al., 2000). The association with C2 deficiency, however, is

less strong, in the order of 10%, whilst C3 deficiency is associated primarily with

membranoproliferative glomerular nephritis (MPGN) (Pickering et al., 2000) with

only two cases of a lupus-like condition being reported. A report showing that a single

nucleotide polymorphism (SNP) in the C1qA gene resulting in low serum levels of

C1q was associated with subacute cutaneous lupus erythematosus provides further

evidence for an association between low C1q and autoimmunity (Racila et al., 2003).

There are currently primarily two favoured mechanisms whereby deficiency or

low levels of the classical complement pathway might lead to the autoimmune

manifestations of SLE, and these are not mutually exclusive. One is the “waste-

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disposal” hypothesis where, in the absence of complement, apoptotic cells and

immune complexes fail to be cleared effectively promoting further autoantibody

production and inflammation. The second, “tolerance” hypothesis, relates to the role of

complement in regulating both B and T cell immune responses (Manderson et al.,

2004). Each of these will be discussed in the context of the particular roles of C1q and

C3.

“Waste Disposal” hypothesis

C1q acting independently of complement activation

We will first consider the ways in which C1q might act in a complement

activation independent manner to influence self tolerance. Rosen and colleagues first

showed that the autoantigens recognised by the lupus autoantibodies, such as

ribonuclear proteins, are localised on the surface blebs of apoptotic cells (Casciola-

Rosen et al., 1994; Rosen et al., 1995). C1q was shown to bind directly to these

surface blebs via its globular head region (Korb and Ahearn, 1997; Navratil et al.,

2001) and to mediate clearance of these dying cell via the C1q receptors calreticulin

(CRT), also known as cC1qR, and CD91 that are expressed on monocytes and

macrophages (Ogden et al., 2001; Vandivier et al., 2002). C1q can also enhance

macrophage-mediated clearance of apoptotic cells by upregulating the expression of

the receptor-ligand pair Mer tyrosine kinase receptor and the growth arrest-specific 6

(Gas6) (Galvan et al., 2012). More recently the scavenger receptor SCARF1

(originally known as ‘scavenger receptor expressed by endothelial cell 1’, SREC-1)

has also been shown to play an important role in the clearance of C1q-coated apoptotic

cells (Ramirez-Ortiz et al., 2013). The binding of C1q to apoptotic cells via its

globular head region enhances uptake of the apoptotic cells by phagocytes, a process

mediated by the exposed C1q-collagen-like tail region (Bohlson et al., 2007)

(Illustrated in Figure 1). This C1q-mediated uptake also actively promotes suppression

of proinflammatory cytokines such as IL1 and IL1 and increases IL10, in

response to LPS stimulation (Benoit et al., 2012; Fraser et al., 2006). Further studies

have highlighted that the interaction of C1q with apoptotic cells is complex and

depends on whether the cells are in early (EAL) or late (LAL) apoptosis. Furthermore

both the rate of ingestion and the modulation of the cytokine response to these C1q-

coated apoptotic cells depend on the differentiation state of the monocytes,

macrophages or DCs (Fraser et al., 2009). Additional evidence for a direct regulatory

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effect came from the observations that incubation of DCs with C1q led to the cells

expressing a predominantly immunosuppressive phenotype as a result of increased

IL10 and reduced IL12 and IL23 synthesis, again in response to LPS activation

(Castellano et al., 2007; Teh et al., 2011; Yamada et al., 2004).

Several signalling pathways have been implicated in this regulation. For

example, it was found that the interaction of C1q with human monocytes resulted in

the induction of inhibitory NF-b p50 homodimer complexes and phosphorylated

cAMP response element binding protein (pCREB) and that these competed with LPS-

mediated proinflammatory signalling pathways (Fraser et al., 2007). Recent findings

demonstrated that in addition to binding directly to DC-SIGN through its globular

(gC1q) region. C1q could also form a trimolecular complex on immature DCs (iDCs)

together with the globular C1q receptor (gC1qR) and DC-SIGN (Hosszu et al., 2012).

Since the gC1qR lacks any transmembrane signalling sequences per se, these results

suggest a possible mechanism whereby C1q/gC1qR could signal through DC-SIGN to

modulate DC activation. This signalling was mediated through the NF-b-p38 MAPK

signalling pathway (Hosszu et al., 2012). Our laboratory has also shown that C1q

plays an important role in DC signalling via a different mechanism. We found that in

C1q-deficient DCs, MAPK p38 and ERK1/2 phosphorylation in response to CD40

signalling was inhibited and this led to a reduced IL12p70 production by these cells

(Baruah et al., 2009). These effects were mediated via the cC1qR (Baruah et al., 2009).

Another possible signalling pathway has been suggested from the discovery that C1q

can bind directly to the inhibitory receptor, leukocyte-associated Ig-like receptor 1

(LAIR-1), on DCs triggering phosphorylation of the two immunoreceptor tyrosine-

based inhibitory motifs (ITIMs) associated with this molecule (Son et al., 2012)

C1q also has been shown to have a direct modulatory role in the production of

the type 1 cytokine interferon-(IFN) by plasmacytoid (p)DCs (Lood et al., 2009;

Santer et al., 2010). IFN is known to be an important factor in the pathogenesis of

SLE (Crow and Kirou, 2004) and has been shown to promote DC maturation (Hardin,

2003; Ronnblom and Alm, 2002). Sera from SLE patients (Vallin et al., 1999),

containing immune complexes of SLE-Ig and material from apoptotic cells such as

small nuclear ribonuclear particles (snRNPs), were able to induce IFNproduction by

pDCs in a TLR 7 and 8 dependent manner (Bave et al., 2000; Lovgren et al., 2004;

Vollmer et al., 2005). When these SLE-immune complexes (SLE-ICs) were incubated

with peripheral blood monocytes (PBMCs) in the absence of C1q they caused

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upregulation of a series of inflammatory interferon response genes similar to those

described in SLE patients. Addition of C1q was able to suppress the expression of

many of these genes (Santer et al., 2012). The mechanism whereby C1q modulates

IFN production by pDCs, however, remains somewhat controversial. It was

originally postulated that C1q interacts directly with both PBMCs and pDCs to inhibit

the production of the proinflammatory cytokines (IL6, IL8 and TNF in response to

IC- or CpG-mediated stimulation (Lood et al., 2009). However, an alternative

mechanism was proposed when it was demonstrated that the role of C1q was to

primarily target uptake of SLE-ICs to monocytes, where they accumulated in the early

endosomal compartment. This preferential targeting to monocytes prevented the

production of IFN by pDCs (Santer et al., 2010). From these observations it is

evident that C1q may play an important role in downregulating IFN production by

activated pDCs and the conflicting findings cited above reflect the complexity and

diverse nature of these effects. Furthermore the fact that C1q and other complement

components are synthesised by macrophages (Petry et al., 2001) and immature

monocyte-derived DCs (Castellano et al., 2004) suggests that its modifying effect(s)

can take place locally without the need for any contribution from plasma-derived C1q.

C1q acting via complement activation: the role of C3 and its activation fragments

As well as acting independently, as outlined above, C1q, as a component of the

C1 complex, is well known to bind to immune complexes, triggering the classical

pathway activation and promoting their solubilisation and clearance (Schifferli et al.,

1985). In humans the clearance of immune complexes is mediated primarily by

erythrocytes expressing the complement receptor 1 (CR1) and results in their transport

to phagocytic cells in the spleen and liver where they are cleared from the circulation

(Cornacoff et al., 1983). SLE patients with lower levels of complement and lower

expression of CR1 on their erythrocytes were found to have impaired splenic immune

complex uptake. This resulted in a transient increase in uptake by the liver and

subsequent release of unbound immune complexes into the circulation where they

could deposit in the tissues thus potentially causing damage (Davies et al., 1992).

C1q can also trigger classical pathway activation following the binding to

phosphatidylserine, annexin A2 and A5, polyclonal IgM or CRP on apoptotic cells

(Kim et al., 2003; Martin et al., 2012; Mevorach et al., 1998; Ogden et al., 2005;

Quartier et al., 2005). This results in C3 activation and C3b/iC3b deposition on the

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apoptotic cells causing their enhanced uptake via complement receptors such as CR3

(CD11b/CD18), CR4 (CD11c/CD18) and CRIg (complement receptor of the

immunoglobulin superfamily) that are expressed on macrophages and DCs. Uptake of

apoptotic cells per se by macrophages suppresses inflammation and actively promotes

tolerance through increased TGF and IL10 and decreased TNF production (Fadok

et al., 1998; McDonald et al., 1999; Voll et al., 1997). Furthermore, additional

signalling by iC3b through CR3 specifically causes downmodulation of DC

stimulatory signals, maintaining an immature DC phenotype that is more likely to lead

to tolerogenic rather than immune-stimulatory responses (Behrens et al., 2007;

Skoberne et al., 2006; Varga et al., 2007; Verbovetski et al., 2002) (Illustrated in

Figure 1). In the absence of C1q and the early classical pathway components,

opsonisation and clearance of dying cells is impaired. This results in the accumulation

of large numbers of apoptotic and necrotic cells expressing self-antigens, that are

available to be taken up by iDCs. The level of activation of these iDCs will determine

the outcome of the response – whether induction of tolerance or immune activation

(Savill et al., 2002). Our previous study using a wide range of murine sera lacking

individual and combinations of classical, lectin and alternative pathway complement

components has highlighted the importance of C3b/iC3b opsonisation in the disposal

of dying cells by macrophages (Quartier et al., 2005). The importance of the role of

C1q acting through activation of the classical pathway has also been suggested by a

recent report describing a patient with normal levels of C1q protein but a homozygous

mutation in the B chain: GlyB63Ser. This mutation did not affect the ability of the C1q

molecule to bind to immune complexes, pentraxins or apoptotic cells but completely

abolished its ability to bind to C1r2C1s2 and form a functional C1 complex

(Roumenina et al., 2011). Since this patient was found to suffer from SLE, this case

suggests that the primary role for C1q is as activator of the classical pathway.

However in this study, not all the known C1q-mediated effect(s) were investigated and

the possibility that C1q may act independently of complement activation cannot be

excluded. In addition one could also speculate that there may be C1q-mediated

protective effect(s) yet to be uncovered.

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Figure 1. C1q: conventional and complement-activation independent effects.

There is now good evidence that the role of C1q is not restricted to complement

activation. C1q can bind directly to apoptotic cells (Apop) promoting their phagocytic

uptake by macrophages (Mac) and DCs resulting in a tolerogenic phenotype. C1q can

also suppress IFN- production by pDCs and T cell proliferation. Furthermore C1q

promotes adhesion and angiogenesis. As component of the classical complement

pathway C1q will lead to C3 cleavage and C3 fragments can contribute to immune

regulation. C3a can promote T cell survival, whilst binding of C3b/iC3b to CR3 on

macrophages and DCs results in increased phagocytosis and tolerance. EC =

endothelial cells

“Tolerance” hypothesis: Regulation of B and T cell responses.

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The other proposed mechanism whereby complement can contribute to disease

susceptibility in SLE relates to its role in regulating the adaptive immune response,

and is known as the “tolerance” hypothesis (Carroll, 2004). It has long been

recognised that complement enhances B cell signalling through co-ligation of the B

cell receptor (BCR) and complement receptor 2 (CR2) expressed on B cells and

follicular DCs, by antigen-bound C3d fragment (Carter and Fearon, 1992; Fearon and

Carter, 1995). During B cell development, a rigorous negative selection of

autoreactive B cells takes place when these cells encounter self-antigens, resulting in

receptor editing, clonal deletion or anergy (Cyster and Goodnow, 1995). A role for

complement in this process was proposed from the findings that negative selection

fails to take place in the absence of C4 or CR1/CR2 (Chatterjee et al., 2013; Prodeus et

al., 1998), suggesting that in the absence of the complement classical pathway,

potentially autoreactive B cells fail to become negatively selected or tolerised (Carroll,

2004). However other factors, such as the genetic background of the mouse strain,

may have also contributed to these findings since it was found, using a similar

transgenic mouse model, that C1q deficiency did not affect B cell tolerance (Cutler et

al., 2001).

C1q acting independently of complement activation

A wide range of T cell abnormalities has been described in SLE patients

(Konya et al., 2014; Moulton and Tsokos, 2011; Tsokos et al., 2003). There is also

emerging evidence that complement contributes to the regulation of T cell responses

(Kemper and Kohl, 2013) and thus may play a role in lupus pathogenesis through such

mechanisms. For example, human T cells expressing gC1qR were found to exhibit

reduced mitogen responses in the presence of C1q (Chen et al., 1994) (illustrated in

Figure 1) and more recently, immobilised C1q was shown to downregulate IL4 and

upregulate IL10 production by human T cells in response to TCR stimulation (Lu et

al., 2007). Furthermore, the effects of C1q on APC function will in turn affect T cell

responses. On the one hand C1q was shown to be required for DC maturation and the

subsequent INF production by T cells (Csomor et al., 2007; Cutler et al., 1998),

whilst on the other hand, it has also been found to downmodulate DC responses

(Fraser et al., 2007; Hosszu et al., 2012; Son et al., 2012; Teh et al., 2011; Yamada et

al., 2004). These apparently contradictory findings could be reconciled by the

observation that C1q regulates monocyte-derived DC generation through the relative

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expression levels of gC1qR and cC1qR on monocytes compared with DCs (Hosszu et

al., 2010). This led to the hypothesis that in the absence of antigen, the interaction of

C1q with gC1qR on monocytes maintains them in a steady state, whilst in the presence

of antigen, C1q undergoes a conformational change that will result in its binding to

cC1qR and in the development of DCs with a more mature phenotype (Ghebrehiwet et

al., 2014). This hypothesis highlights the complexity of the signalling pathways

triggered by C1q on different receptors and the diversity of functions mediated by this

molecule. In this context it is worth mentioning that in addition to regulating immune

responses, C1q has recently been shown to be involved in a variety of other important

biological functions including fibroblast and endothelial cell adhesion (Ghebrehiwet et

al., 2012), trophoblast migration (Agostinis et al., 2010) and more recently in

angiogenesis and wound healing (Bossi et al., 2014)

C1q acting via complement activation: the role of C3 and its activation fragments

Activation of the classical pathway via C1q leads to formation of C3 activation

fragments. There is evidence that C3 contributes to both CD4+ and CD8+ T cell

priming and to allogeneic-B and -T cell responses (Kopf et al., 2002; Marsh et al.,

2001; Suresh et al., 2003). Furthermore, the complement anaphylotoxin fragments C3a

and C5a, by signalling though their respective receptors, C3aR and C5aR, have been

shown to provide costimulatory, survival and anti-apoptotic signals to T cells directly

(Lalli et al., 2008; Strainic et al., 2008) and indirectly via DC activation (Lalli et al.,

2007; Li et al., 2008; Peng et al., 2008). Since C3a and C5a fragments are generated

via activation of the alternative pathway, and FB and FD were found to be produced

following T cell activation by APCs presenting their cognate peptide (Heeger et al.,

2005), these findings have led to the hypothesis that local immune cell-derived

complement is important in the induction of T cell responses. In this context it is worth

noticing that components of the coagulation system such as thrombin and plasmin can

also cleave C3 and C5 to generate the respective activation fragments (Amara et al.,

2008))., Furthermore a recent report showing that C3 can be cleaved by intracellular

cathepsin L suggests that C3 activation can also take place within the cell

independently of other complement components. This intracellular C3 cleavage

appears to have a key role in T cell homeostasis (Liszewski et al., 2013).

Consistent with the notion that complement can regulate adaptive immunity,

C3 has been shown to play a role in the suppression of contact sensitisation, the

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generation of oral tolerance and more recently the differentiation of myeloid

suppressor cells (Hammerberg et al., 1998; Hsieh et al., 2013; Pekkarinen et al., 2013;

Purwar et al., 2011). Therefore there is conflicting evidence in the literature and the

role of C3 appears to be paradoxical. In contrast to the report that C3 is required for

the rejection of fully MHC mis-matched kidney grafts (Pratt et al., 2002), we have

shown, using the minor histocompatibility (HY) model of skin graft rejection, that

C1q and C3, are required for the induction of tolerance rather than for rejection

(Baruah et al., 2010), a finding that was subsequently confirmed by others (Bartel et

al., 2013). Using C1q-, C3-, C4- and C5-deficient female mice we have also

demonstrated that the classical pathway components including C3 are required for

tolerance induction, whilst C5 plays no role (Fossati-Jimack et al., 2015). Of note, the

C3-deficient mice used in our study cannot generate any intracellular C3a fragment as

they lack the corresponding genetic region (Wessels et al., 1995). In addition recent

studies have called into question whether C5aR is expressed on murine CD4+ T cells

(Dunkelberger et al., 2012; Karsten et al., 2014). These results would therefore argue

against the notion that C3a and C5a signalling through their respective receptors is

important for T cell activation, and that the absence of this signalling leads to Treg

generation, as suggested by a recent report (Strainic et al., 2013). Rather we found

that C3 is required for the establishment of the Treg-DC tolerogenic loop critical for

the induction and maintenance of tolerance (Min et al., 2003; Yates et al., 2007). We

also found no evidence of a defect in the ability of C3-deficient T cells to generate

Treg using established in vitro protocols, arguing against an intrinsic T cell defect.

We did, however, identify a role for C3 in the induction of the amino acid

metabolizing enzyme, nitric oxide-synthase-2 (Nos-2) (Fossati-Jimack et al., 2015),

which has been shown to be crucial for Treg generation in a model of infectious

tolerance (Cobbold et al., 2009). Thus, in the absence of C3, the induction of amino

acid-depleting enzymes in DCs is impaired causing a reduced Treg generation

possibly as a result of impaired downregulation of the mTOR pathway. Interestingly,

these effects might be mediated by iC3b acting through CR3 (Behrens et al., 2007;

Skoberne et al., 2006; Varga et al., 2007; Verbovetski et al., 2002) since we have

additionally found that signalling by iC3b-coated particles through CD11b/CD18 on

bone marrow-derived DCs (BMDCs) led to downregulation of MHC Class II and

CD86 expression. These effects were dependent on CD11b expression (Figure 2).

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Figure 2. Ligation of CD11b/CD18 on DCs with iC3b-coated red blood cells (iC3b-RBCs) impairs lipopolysaccharide (LPS) and CD40 mediated upregulation of MHCII and CD86. Murine BMDCs were incubated with medium alone, guinea pig RBCs or iC3b-RBCs for 1 hour followed by stimulation with LPS (100ng/ml) or CD40L plus enhancer (500ng/ml and 1g/ml respectively). After 24 hours the cells (live-cell gated) were analysed for expression of MHCII and CD86 by flow cytometry. (A) B6 WT DCs (B) CD11b deficient (B6.Itgam-/-) BMDCs. (C) DCs stimulated with LPS, (D) DCs stimulated with CD40L and enhancer. Results are expressed as percentage of change of MHCII and CD86 expression in DC pre-incubated with iC3b-RBC compared to DC pre-incubated with RBC alone. Data from 6 independent experiments; the bars represent mean +/- SEM. The dotted line indicates zero change, where the values for pre-incubation with iC3b-RBC and RBC are identical.

C3 and autoimmunity - a unifying hypothesis.

As outlined above, both C1q, acting independently of its role in classical

pathway activation, and C3, following activation by the classical, lectin or alternative

pathways, have important roles in the clearance of apoptotic cells and immune

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complexes. In addition both can exert either activating or downmodulating roles on T

cell responses directly through receptor-mediated signalling pathways (Chen et al.,

1994; Csomor et al., 2007; Cutler et al., 1998; Lu et al., 2007; Peng et al., 2008;

Strainic et al., 2008) and indirectly through DC modulation (Behrens et al., 2007;

Csomor et al., 2007; Fraser et al., 2007; Hosszu et al., 2012; Peng et al., 2008;

Skoberne et al., 2006; Son et al., 2012; Teh et al., 2011; Varga et al., 2007;

Verbovetski et al., 2002; Yamada et al., 2004). Since both molecules perform such

apparently analogous roles, it is somewhat perplexing that deficiencies in C1q and

classical pathway components (C4, C1r and C1s) predispose to autoimmunity, whilst

C3 deficiency does not. A possible explanation for this discrepancy comes from our

recent findings that C3 has an additional role: it can influence intracellular trafficking

by inducing delayed lysosomal fusion of apoptotic cell cargo (Baudino et al., 2014).

This limits the endocytic processing of antigens, that is necessary for optimised T cell

antigen presentation (Delamarre et al., 2005). We found that in the absence of C3, but

not of C1q, phagosome maturation is accelerated resulting in reduced T cell responses

(Baudino et al., 2014). Our findings are thus in agreement with previous observations,

made in B cells, that covalently bound C3b can protect an antigen from degradation

within the endosomal compartment, again enhancing MHC class II antigen

presentation (Jacquier-Sarlin et al., 1995; Serra et al., 1997). In this context the role of

C3 is similar to the one recently reported for other opsonins and receptors, such as

MFG-E8 and DNGR-1, that can direct the trafficking and subsequent cross

presentation of antigens from dying cargo (Peng and Elkon, 2011; Zelenay et al.,

2012). Our recent results therefore demonstrate that another potential mechanism by

which C3 deficiency may protect from the development of autoimmunity is by

reducing the T cell response to self-antigens displayed on dying cells. Hence C3 is

uniquely required for both the generation of optimal T cell immune responses and for

the clearance of the autoantigens on the apoptotic cargo. In the absence of C3, both

arms of this process are impaired and autoimmunity is prevented.

Conclusions

As outlined in this review, complement has a key role in the development and

pathology of autoimmune conditions, especially SLE. Over the last decades it has

become apparent that C1q, in addition to its traditional role as classical pathway

initiator, can act independently of complement activation. This large number of C1q-

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mediated functions offers a plethora of possible explanations for the observations that

C1q deficiency predisposes to the development of SLE. Despite the fact that none of

the current models provides a unifying mechanism for all the findings, the observation

that C3 deficiency does not predispose to autoimmunity can perhaps now be

explained. C3 opsonisation can regulate the endocytic handling of apoptotic cells, a

process that is essential for optimal T cell activation. In the absence of C3, the T cell

proliferation in response to apoptotic cell-associated self-antigens is impaired and this

may prevent the development of SLE.

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