2004 Nature Publishing Group http://www.nature.

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REVIEW

The complement system in regulation

of adaptive immunity
Michael C Carroll
The serum complement system, which represents a chief component of innate immunity, not only participates in inflammation
but also acts to enhance the adaptive immune response. Specific activation of complement via innate recognition proteins or
secreted antibody releases cleavage products that interact with a wide range of cell surface receptors found on myeloid, lymphoid
and stromal cells. This intricate interaction among complement activation products and cell surface receptors provides a basis
for the regulation of both B and T cell responses. This review highlights fundamental events, explaining how complement links
innate and adaptive immunity as well as describing more recent studies on how this large family of proteins functions locally in
peripheral lymph nodes to enhance B and T cell responses.

The complement system was first identified as a heat-sensitive factor

in fresh serum that complemented the effects of specific antibody in
the lysis of bacteria and red blood cells1. Its importance as an effector
of humoral immunity was extended with the early observations of
opsonization and participation in cellular immunity2. From these
early studies demonstrating its importance in host protection, the
complement system was considered a chief component of innate
immunity. It is now appreciated that this system represents a complex
pathway of more than 30 serum proteins and cell surface receptors
that interact in a range of functions from direct cell lysis to the
enhancement of B and T cell responses3,4. Like the more recently
identified Toll-like receptors (TLRs), the complement system can be
activated by hard-wired pattern-recognition receptors that have
evolved to recognize pathogen-associated molecular patterns
(PAMPs)5,6. Most recognition structures involved in complement
activation are serum proteins that, in addition to specific antibody,
include pattern-recognition receptors such as mannan-binding lectin
(MBL)7, ficolins8, C-reactive protein9, C1q10,11 and natural
immunoglobulin M (IgM)1214.
Complement is activated by three different pathways: classical,
lectin and alternative. All three share the common step of activating
the central component C3, but they differ according to the nature of
recognition. The classical pathway was the first studied; it is activated
by antibody released after a humoral response or by natural antibody
(discussed in more detail below)3. The lectin pathway is activated
after the recognition and binding of PAMPs by lectin proteins. To
date, three members of this pathway have been identified: MBL15,
ficolin H and ficolin L16. MBL is a C-type lectin and a member of the

collectin family of proteins that includes both collagen and globular

regions. Structurally MBL is similar to C1q. However, the six globular heads of MBL form carbohydrate-recognition domains and bind
N-actetyl glucosamine and mannose, which are common among
microbes. Given the nature of the carbohydrate recognition
domains, this pathway is probably the oldest in evolutionary terms.
Ficolins are also lectin-type recognition proteins and, like MBL, they
recognize N-actetyl glucosamine and mannose structures and
include both collagen and globular regions. Both MBL and ficolins
are associated with serine proteases or MBL-associated serine proteases, which act like those members of the classical pathway in activating components C2 and C4 leading to the central C3 step16,17.
Finally, the alternative pathway contrasts with both the classical and
lectin pathways in that it is continuously turned on because of the
spontaneous activation of C3 and its promiscuity in binding to a
wide range of suitable acceptor sites18,19.
Given the multiple pathways of activation and the catalytic nature
of the many steps, regulation of the complement system is complex.
To limit host destruction, the system makes use of both serum and
cell surface regulatory proteins. Almost all mammalian cells express
regulators of complement to protect against an autologous attack on
self20. The finding that some of these regulators of complement also
participate in adaptive immunity led to the general idea of Fearon and
Carter that the complement system links innate and adaptive immunity21. It was proposed that the complement system instructs the
humoral response in a way that is similar to that of the direction of the
CD4+ T cell response by dendritic cells (DCs)22. This review expands
on this idea of how the complement system is intricately involved in
regulating not only humoral but T cell immunity.

The CBR Institute for Biomedical Research, Harvard Medical School, 800
Huntington Ave., Boston, Massachusetts 02115, USA. Correspondence should
be addressed to M.C.C. (carroll@cbr.med.harvard.edu).

Regulation of humoral immunity

Evidence that the complement system was involved in adaptive
immunity can be traced over three decades to the early reports of
binding of C3 to circulating lymphocytes23 and follicular dendritic

Published online 28 September 2004; doi:10.1038/ni1113

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REVIEW
cells (FDCs) within lymphoid follicles24. These findings, combined
with experimental results showing that a transient reduction in the
amount of circulating complement C3 led to an impairment in antibody response, suggested that the complement system was involved in
adaptive immunity25.
A fundamental event following activation of the complement system is covalent attachment of C3b to the foreign (or self) antigen26,27.
Conceptually, this is a key event in innate immunity because it provides a mechanism to mark or identify antigens (both foreign and
self) for uptake by phagocytic cells or retention on FDCs for recognition by cognate B lymphocytes (discussed in more detail below). Both
C3 and C4 components have an internal thioester that upon activation can form amide or ester linkage28. This usually follows proteolytic cleavage via the classical or lectin pathways. However, as
mentioned above, C3 can spontaneously form a covalent linkage and
provide a substrate for amplification via the alternative pathway29.
Complement enhances B cell immunity principally via complement receptors CD21 (which binds iC3b, C3d,g and C3d) and CD35
(which binds the same C3 products as CD21 but also binds C3b and
C4b)30. In mice, the two receptors seem to be coexpressed and represent splice products of a single locus (the Cr2 locus)31,32. They are
expressed mainly on B cells and FDCs. On B cells, CD21 forms a
receptor complex with the signaling protein CD19 and tetraspan
protein CD81 (ref. 33). Uptake of C3d-coated antigen by cognate B
cells results in an enhanced signal via the B cell antigen receptor34.
Thus, coengagement of the CD21-CD19-CD81 coreceptor with B
cell antigen receptor lowers the threshold of B cell activation and
provides an important survival signal. A second key mechanism by
which complement enhances B cell immunity is by localization of
antigen to FDCs within lymphoid follicles35. FDCs are specialized
stromal cells that secrete the B lymphocyte chemoattractant
chemokine36 and are important in organizing germinal centers

within the B cell follicles37. FDCs have relatively high expression of

CD21 and CD35, and this provides an effective mechanism for
retention of C3-coated immune complexes within the lymphoid
compartment. How immune complexes are taken up by FDCs is not
clear, but an intact classical pathway and CD21 and CD35 receptors
are thought to be essential38.
Studies in mice bearing targeted deficiencies of complement C1q39,
C4 or C3 (ref. 40) or CD21-CD35 receptors41,42 demonstrate the
involvement of complement in multiple stages of B cell differentiation43 (Fig. 1). B cells first express the CD21-CD19-CD81 coreceptor
at the transitional stage as they migrate from the bone marrow into the
periphery44. This stage of development is important in the elimination
of self-reactive B cells because crosslinking of the B cell antigen receptor results in cell death or anergy rather than activation45. Although
direct involvement of the CD21-CD19-CD81 coreceptor at this stage is
not apparent, coligation with the B cell antigen receptor by C3- or C4coated self-antigens could lead to enhanced negative signaling.
All mature B cells have varying expression of CD21 and CD35. For
example, follicular B cells and peritoneal B1 cells express low to
intermediate amounts, whereas marginal zone B cells express relatively high amounts, which they use to transport C3-coated immune
complexes from the splenic marginal zone into the follicles46,47 (discussed more below). The importance of this difference is not apparent, but functional involvement of the coreceptor in each
subpopulation has been suggested (Fig. 1). B1 cells, which are the
chief source of natural antibody, are positively selected during early
development and are thought to be self-replenishing, unlike conventional B cells4850. Complement seems to function in the selection or
maintenance of B1 cells, as mice deficient in CD21 and CD35 receptors have an altered repertoire51,52.
Activation of naive mature B cells is enhanced by the presence of an
intact classical pathway, as mice deficient in early pathway components (C1q, C4 or C3) have impaired
humoral responses to thymus-dependent
and thymus-independent antigens4. Notably,
Spleen, bone marrow
mice deficient in CD21 and CD35 receptors
4
have a similar impairment suggesting that
overall enhancement by complement is
mediated by these receptors41,42. However, as
discussed below, C3 ligands can also affect
Memory B
adaptive immunity by other pathways.

Peripheral compartment
Transitional

Mature

Activation

1
B1

B2

B2

Anergy or
apoptosis

Plasma cell

MHC peptide

C3d

Antigen

CD21, CD19

B1

B2

CD35

FDC

Figure 1 Complement receptors are important in the regulation of B lymphocyte differentiation at five
stages. Stage 1: B1 cells represent a subset of B cells that preferentially develop during early life and
are positively selected by self or microbial antigens, probably at the transitional stage. CD21-CD19CD81 coreceptor engagement by complement-coated antigens enhances positive selection. Stage 2:
Self-reactive B cells are eliminated in the bone marrow and at the peripheral transitional stage.
Coligation of the B cell antigen receptor (BCR) and CD21-CD19-CD81 coreceptor by C3- or C4-coated
self antigen alters negative signaling. Stage 3: Activation of naive mature follicular B cells by the
engagement of antigen coupled to complement C3d results in coligation of the B cell antigen receptor
and coreceptor; in the presence of T cell help, this leads to activation and expansion. Stage 4:
Activated B cells initiate the formation of a germinal center in the splenic follicles, which is organized
by FDCs. Complement receptors expressed on germinal center B cells enhance BCR signaling. In
addition, complement receptors expressed on FDCs retain antigen and promote the antigen selection
of high-affinity germinal center B cells. Stage 5: Post-GC B cells require complement and antigen
selection for the efficient maintenance of long-term memory B cells.

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Regulation of T cell immunity

Janeways conceptualization of the adjuvant
effect as being due to the influence of the
innate immune system on T cell immunity
led to an intense study of how pathogens are
recognized by DCs and other professional
antigen-presenting cells (APCs)53. Much of
the focus has been directed at the growing
family of TLRs that recognize a wide range of
bacterial structures54. Not unexpectedly, the
complement system also seems to participate
in T cell immunity through its property of
marking antigens as foreign and recognition
by specific receptors. Early studies examining
the function of complement in T cell priming
suggested that its involvement was more
important in B cell immunity (as discussed
above). C3-deficient mice immunized with
noninfectious antigens such as haptenated

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REVIEW
keyhole limpet hemocyanin or bacteriophage40,55 developed apparently normal T cell responses, yet humoral responses were impaired.
Likewise, initial work with infectious herpes simplex virus or inactivated lymphocytic choriomeningitis virus in C3-deficient mice found
no alteration in the priming of CD4+ T cells, despite the impairment
of B cell immunity56,57. In addition, impairment of B cell responses by
blocking of Cr2 receptors did not alter priming of T helper cells58.
A report that the priming of both CD4+ and CD8+ T cells was
impaired in C3-deficient mice in an influenza virus model supports
the idea of more general involvement of the complement system in
adaptive immunity59. Although the mechanism remains unclear, C3
is required in the pulmonary T cell response to influenza. Challenge
of Cr2-deficient mice in a similar model indicates that T cell priming
is normal, suggesting that the CD21-CD19-CD81 B cell coreceptor is
not involved. One possible explanation is that APCs preferentially
take up C3-coated viral particles for the priming CD4+ and CD8+ T
cells via complement receptors such as CR3 (CD11b-CD18) or CR4
(CD11c-CD18). In the absence of C3, APC function could be
reduced, resulting in a limited priming of T cells. An alternative possibility that is not mutually exclusive of an APC defect is that the
chemokine-like C3a and C5a receptors are important in the pulmonary response.
A fundamental event following the activation of complement C3
and C5 is release of N-terminal peptides of approximately 9 kilodaltons. They are byproducts of all three complement activation pathways and serve as potent ligands for G proteincoupled
chemoattractant receptors referred to as C3aR and C5aR, respectively6062. They are expressed on a wide range of inflammatory cells,
such as mast cells, eosinophils, basophils and lymphocytes, as well as
smooth muscle cells. They seem to participate in inflammatory events
via direct cell activation and in the modulation of cytokine release by
macrophages and/or monocytes. In the influenza model, the absence
of C3a and C5a ligands could influence the infiltration of CD4+ and
CD8+ T cells, as is described in more detail below.
How complement is activated in the influenza model is not
known, but one strong possibility is that specific natural antibody
binds virus particles and activates complement via the classical
pathway. Studies showing involvement of natural antibody in the
humoral response to influenza support this possibility63. Further
support for the idea of involvement of natural IgM and complement
in priming CD8+ T cells comes from a leishmania recombinant antigen hydrophilic acylated surface protein B1 (HASPB-1) model64.
The investigators proposed a pathway in which the recognition of
HASPB-1 by natural IgM activates classical pathway complement,
leading to the production of interleukin 4 (IL-4) by a CD11b+
CD11clo population of macrophages and the stimulation of IL-12
release by DCs. How complement activation stimulates macrophage
release of IL-4 is not clear. One possibility is that it occurs by direct
opsonization via the CR3 receptor with C3-coated antigen.
Although crosslinking CR3 is not thought to stimulate the release of
IL-4 in general, coligation with a HASPB-specific TLR could provide a suitable stimulus.
Natural antibody and classical pathway complement also seem to
be involved in contact sensitivity. In a complex model, involving
natural killer T cell release of IL-4 and the stimulation of B1 cell
production of natural IgM, Askenase and colleagues observed that
the early stage of contact sensitivity is complement-dependent65.
Notably, complement is activated via the innate natural IgM pathway, similar to that of the HASPB-1 model discussed above66.
However, in the example of contact sensitivity, IL-4 production lies
upstream of B1 cell release of IgM.

NATURE IMMUNOLOGY VOLUME 5 NUMBER 10 OCTOBER 2004

In an elegant series of experiments, Wetsel and colleagues first

demonstrated a role for C3 in a mouse model of asthma. They found
that mice deficient in C3 were partially protected against airway
hyper-reactivity, eosinophil infiltration and IL-4 production after
multiple challenges with a mixture of Aspergillus fumigatus (cell culture filtrate) and ovalbumin67. The response is characterized in normal mice by the infiltration of eosinophils, production of IL-4 by
antigen-specific T cells and airway hyper-reactivity. Thus, this provided strong support for the idea of involvement of C3 in T helper
type 2dependent CD4+ T cell model of asthma. In later studies, the
same group identified a similar protective phenotype in mice bearing
a targeted deficiency of C3aR68. This suggests that release of C3a, via
either direct cleavage by bacterial products or activation of one of the
three complement pathways, leads to triggering of the C3aR and stimulation of cytokine release. The mechanism in vivo is not clear but as
both APCs and T cells express C3aR, the results support the idea of
involvement of C3aR in the linkage of innate and adaptive immunity.
It will be useful to determine whether C3a release in this model is
dependent on natural antibody, as found in the leishmania antigen
study discussed above.
The other complement-related chemoattractant receptor, C5aR, is
also potentially involved in the regulation of T cell responses in the
lung. Mice bearing a targeted deficiency in C5aR were first shown to
have a reduced response to pulmonary infections with Pseudomonas
aeruginosa69. Although infiltration of neutrophils seemed normal in
the infected animals, clearance was substantially impaired in the lung.
Notably, neutrophil clearance was lung specific, as infection in the
peritoneum was cleared by the C5aR-deficient mice. The C5a ligand
and its receptor seem to have a regulatory function in human and
murine asthma. Karp and colleagues mapped susceptibility to allergen-induced airway hyper-reactivity in humans to the C5 locus70.
They proposed that C5 ligand release induces IL-12 production,
which has a dampening effect on the T helper type 2dependent airway hyper-reactivity. However, the model is complex: triggering of
C5aR also downregulates IL-12 (ref. 71).
The activation of T cells is tightly regulated and mechanisms have
been identified both in the thymus (central tolerance) and in the
periphery. Control of activated CD4+ T cells in the periphery is mediated by both specific elimination and anergy. Recent observations
support the idea of a third mechanism: that of regulatory T cells. The
complement system seems to participate in development of human
regulatory T cells via costimulation of CD3 and CD46. A member of
the complement regulator receptor family, CD46 (membrane cofactor protein), is associated with this. Early studies had demonstrated
that crosslinking of CD46 (which is also the measles virus receptor)
on human monocytes led to an impairment in the expression of IL12, which is necessary for cell-mediated immunity in measles infection72. It was proposed that uptake of C3b-opsonized virus via CD46
could block IL-12 and that this might explain the T cell suppression
observed in humans after an infection with measles virus. Later
experiments supported the idea of an alternative function for CD46.
Kemper et al. found that crosslinking of CD3 and CD46 on human
CD4+ T cells led to the induction of a regulatory T phenotype and
release of IL-10 (ref. 73). The induced regulatory T cells proliferated
in culture, blocked activation of bystander T cells and differentiated
into memory cells. Thus, these findings support the idea of a previously unknown function for complement in the differentiation of
regulatory T cells.
In summary, it is becoming increasingly clear that the complement system participates in regulation of T cells by multiple mechanisms such as direct opsonization of foreign antigens by APCs and

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2 10 PFU
HSV-I
DC
1

DC

2
DC

Afferent
lymph

B B

2004 Nature Publishing Group http://www.nature.com/natureimmunology

7
B

DC

Lymph
node

B B

M
5

MHC
class II

C3

IFN-

Viral
antigen

Antibody

T cell
receptor

Phagocytic
cells

Figure 2 Complement enhances B cell response to herpes simplex virus 1

introduced intradermally. Step 1: Intradermal infection with herpes
simplex virus 1 (HSV-1) induces inflammation, promoting edema and
leakage of serum proteins into dermis. Complement is activated and binds
to viral antigens. Infiltrating leukocytes are also a potential source of C3
for binding to viral antigens. Steps 2 and 3: DCs take up viral antigen and
migrate into local (peripheral) lymph nodes for delivery to T cells. Viral
antigen passively drains into the peripheral lymph nodes. Steps 4 and 5:
Activated HSV-1-specific T cells release cytokines such as interferon-
(IFN-), which stimulate macrophages to release early classical pathway
complement proteins Step 6: Recognition of viral antigens by natural or
immune IgM activates complement and C3 attaches to antigen. Steps 7
and 8: Cognate B cells respond to C3-coated antigen via coligation of the
B cell receptor and coreceptor, expanding into antibody-forming cells and
the formation of a germinal center.

by modulating cytokine release. Stimulation of the chemoattractant

receptors C3aR and C5aR by products of complement activation also
alters T cell responses by mechanisms that remain unclear. Finally, the
CD46 receptor that is widely expressed on human cells appears to be
involved in regulation of the activity of T cells via both cytokine modulation and differentiation of regulatory T cells.
Sources of complement
Consistent with its involvement in systemic immunity, complement
proteins for the most part are synthesized in the liver, where they are
taken into circulation. Although synthesis is principally constitutive,
during infections the release of IL-6 induces upregulation of the acute
phase proteins, which includes certain components such as C3.
Notably, the early components of the classical pathway, C1, C2, C4
and C3, are also expressed by macrophages7476. In particular, proinflammatory cytokines such as IL-6, tumor necrosis factor and interferon- stimulate macrophages to express the individual components.
An explanation for regulated expression is that within peripheral
lymph nodes and local tissues such as the skin, macrophages could
provide a complete source of components for assembly of activated
C3 and coating of foreign (or self-) antigens.
Systemic versus local complement synthesis
To determine the relative importance of C3 and C4 production by bone
marrowderived cells, Verschoor and colleagues prepared radiation
chimeras. In these chimeras, stromal and liver cells were C3 deficient
(or C4 deficient), but bone marrowderived cells were complement
sufficient (normal chimeras) or vice versa; that is, systemic C3 but bone

984

marrowderived C3 deficient (reverse chimeras). Both sets of chimeras

responded to immunogens, both inert and infectious, when challenged
intravenously55,7779. Thus, despite there being negligible amounts of
C3 (or C4) in circulation, normal chimeras responded to immunization with T celldependent antigens with near-normal antibody
response. These results suggested that local production of classical
pathway C4 or C3 was sufficient to provide an enhancing effect to
humoral immunity. The results were further interpreted to suggest that
the critical site for complement synthesis was the secondary lymphoid
tissue as complement deposition (and antigen) was observed on FDCs
in the spleen (for intravenous injection) or draining lymph nodes (for
peripheral injection).
Using the same infectious antigen mode described above (herpes
simplex virus), mice with impaired local production of complement
(reverse chimeras) but normal levels of complement in circulation
respond to intravenous immunization, confirming that serum complement proteins are sufficient for enhancement of systemic immune
responses79. These results were expected because antigens administered intravenously drain into the spleen, where they have contact
with serum complement proteins. Activation and coupling of C3 to
antigens injected intravenously facilitates their uptake on marginal
zone B cells, which participate in transport of antigen into the splenic
follicles46,47,80. In contrast, reverse chimeras show an impaired
response to a herpes viral infection introduced intradermally79. This
finding was somewhat unexpected, because the chimeric animals had
normal amounts of C3 in their circulation yet failed to develop a normal humoral response. Histological examination of the site of viral
infection identified a vigorous infiltration of monocytic cells similar
to that found in wild-type control mice. Moreover, leakage of C3 into
infected tissues was similar in both reverse chimeras and wild-type
mice, suggesting that serum-derived complement was not limiting at
the site of infection. Yet the humoral response to the virus was
impaired. One explanation for the findings is that the critical site for
complement enhancement of B cell immunity is in the draining
peripheral lymph nodes, where activated macrophages produce relatively large amounts of early classical pathway components (Fig. 2).
These findings raise questions regarding the trafficking of viral antigens into the peripheral lymph nodes and encounter with cognate B
cells. Complement is not limiting at the site of infection because of
leakage of serum proteins; therefore, if transport of viral antigens into
the peripheral lymph nodes is complement dependent, it is unlikely
that the macrophage components are critical at this step. It seems
more likely that the limiting step in the function of complement lies
in the peripheral lymph nodes, where a critical threshold for complement levels is required for efficient coupling of activated C3 to antigen and the enhancement of adaptive immunity.
Future studies
Over the past decade our understanding of how the complement
system regulates B cell immunity has greatly expanded. Yet as mechanisms are identified, new questions arise. For example, it is not
apparent how B cells encounter cognate antigens in either primary
or secondary responses. Localization of antigens on FDCs in a complement-dependent way after a primary response suggests that
primed B cells encounter antigen in the follicular zone of secondary
lymphoid tissues, but how antigen is taken up on FDCs remains a
mystery. Another important topic over the next decade will no
doubt focus on unraveling the mechanisms involved in the clearance of self antigens, especially lupus antigens. The finding that
complement deficiencies are chief susceptibility factors for diseases
like lupus suggest it is important in clearance. With technological

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REVIEW
advances in real-time imaging and the construction of conditional
knock-in mice, many of these types of questions can be answered.
Finally, the observations that complement in concert with natural
antibody participates in regulating T cell immunity, both in activation and in control, provides a rich area for further investigation. It is
likely that the complement receptor pathways intersect with other
innate pathways such as the TLRs, as might occur in response to
highly conserved microbial antigens or PAMPs. This topic provides
additional opportunities for exploration.
In summary, the complement system represents a complex component of innate immunity that has many functions, including the
regulation of adaptive immunity. Further study will no doubt lead
to the identification of additional pathways that link innate and
adaptive immunity.
ACKNOWLEDGMENTS
I thank R. Barrington for review of the manuscript, and current and past members
of the laboratory for their contributions to the understanding of how complement
influences adaptive immunity. The related work from my laboratory is supported
by grants from the National Institutes of Health (AI39246-09, AI36389-08,
AI52343-03 and AI53570-02).
COMPETING INTERESTS STATEMENT
The author declares that he has no competing financial interests.
Published online at http://www.nature.com/natureimmunology/
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