Complement-Dependent Transport of
Antigen into B Cell Follicles
This information is current as
of July 31, 2017.
Santiago F. Gonzalez, Veronika Lukacs-Kornek, Michael P.
Kuligowski, Lisa A. Pitcher, Søren E. Degn, Shannon J.
Turley and Michael C. Carroll
J Immunol 2010; 185:2659-2664; ;
doi: 10.4049/jimmunol.1000522
http://www.jimmunol.org/content/185/5/2659
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References
Complement-Dependent Transport of Antigen into B Cell
Follicles
Santiago F. Gonzalez,*,† Veronika Lukacs-Kornek,†,‡ Michael P. Kuligowski,*,†
Lisa A. Pitcher,*,† Søren E. Degn,*,†,x Shannon J. Turley,†,‡ and Michael C. Carroll*,†,{
P
eripheral lymph nodes (LNs) and the spleen make up the
secondary lymphoid organ tissue that provide a specialized environment for circulating B and T lymphocytes
to interact and encounter cognate Ag (1). Although T cells
home to the paracortical region of LNs, B cells traffic to the
follicles in search of Ag. This directed migration is dependent
on chemokines produced by stromal cells in the respective
compartments. Recent elegant intravital imaging of T and
B cell trafficking within the peripheral LNs revealed a directed
migration along stromal “highways” (2, 3). Fibroblast reticular
cells (FRCs) secrete the collagen-rich fibers that form the network within the paracortical region, and they secrete T cell
chemokines CCL19 and CCL21. B cell migration within the
follicles is dependent on follicular dendritic cells (FDCs) dendritic processes and a less-dense network of FRC fibers.
Although the reticular network within LNs was characterized
more than three decades ago (4), only more recently did it
become apparent that it acts as a conduit for the delivery of
cytokines, chemokines, and small-protein Ags to the T (5–8)
and B cell areas (9, 10). B cell conduits are structurally and
*The Immune Disease Institute and Program in Cellular and Molecular Medicine,
Children’s Hospital, †Department of Pathology, and {Department of Pediatrics, Harvard Medical School; ‡Department of Cancer Immunology and AIDS, Dana Farber
Cancer Institute, Boston, MA; and xDepartment of Medical Microbiology and Immunology, Århus University, Århus, Denmark
Received for publication April 12, 2010. Accepted for publication May 19, 2010.
This work was supported by National Institutes of Health Grants 5 RO1 AI039246, 1 PO1
AI078897, 5 RO1 AI067706 (to M.C.C.), and RO1 DK074500 (to S.J.T.); a Glaxo-Smith
Kline postdoctoral fellowship award (to M.K.); Marie Curie International Outgoing Fellowship for Career Development Award 220044 (to S.F.G.); and National Institutes of
Health Transfusion Biology and Medicine Training Grant 5T32HL066987-09 (to L.A.P.).
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000522
immunochemically similar to those in the T cell area. They
differ primarily by specificity of the chemokine secreted (i.e.,
follicular FRCs secrete CXCL-13, whereas paracortical FRCs
secrete CCL19 and CCL21). Although the outer diameter of
conduits is ∼1–2 mm, they are tightly packed with collagen
fibers with a spacing of 5–8 nm, which acts as a molecular sieve
(Fig. 1). Thus, only proteins less than ∼60 kDa enter the
conduits. Whether conduit structures are altered to accommodate larger Ags during infection is not clear.
Trafficking of lymph-borne Ags into B cell follicles
Small protein Ags gain direct access into the B cell follicles via
gaps in the subcapsular sinus (SCS) floor (11) or through the
FRC conduits (Fig. 2A) (9). The latter pathway provides a
directed flow of small Ags to the FDCs for transient retention,
or in the presence of Ab and complement, long-term binding
via specific receptors. Although cognate B cells can access Ag
draining via the conduits (9), their principal role is more likely
directing the Ag to FDCs for stable retention. Although these
initial experiments involved model Ags, such as lysozyme (9),
or OVA (10), in the natural setting it seems likely that a major
source of Ag is degraded products of pathogens that drain from
tissues via the lymphatics, as suggested by Jenkins and colleagues (12).
Lymph-borne particulate Ags, such as vesicular stomatitis
virus (13), and protein-coated beads (14) are rapidly taken up
by macrophages that line the SCS (SSMs) (15). Interestingly,
the particulate Ags are shuttled to the underlying surface where
they are made available to cognate B cells. Similarly, large
protein Ags injected s.c. into passively immunized mice also
seem to bind rapidly to SSMs. However, in the latter example,
capture by SSMs is complement dependent. Thus, formation of
immune complexes (ICs) activates complement, resulting in
formation of C3-coated ICs (C3-ICs) that enhance uptake via
CR3 (Mac-1) and FcRIIb on the SSMs (16). Subsequently,
C3-ICs are relayed to the underlying B cell compartment where
they are transferred to naive B cells (Fig. 2A) (17). FcRIIb is
known to recycle to the surface following internalization and
Address correspondence and reprint requests to Dr. Michael C. Carroll, Immune Disease
Institute, 200 Longwood Avenue, WAB, Room 251, Boston, MA 02115. E-mail address:
[email protected]
Abbreviations used in this paper: C3-IC, C3-coated immune complex; CR, cysteinerich; DC, dendritic cell; FDC, follicular dendritic cell; FRC, fibroblast reticular cell; GC,
germinal center; IC, immune complex; LN, lymph node; MF, macrophage; MBL,
mannose-binding lectins; MM, medullary macrophage; MP-IVM, multiphoton intravital microscopy; MZ, marginal zone; SCS, subcapsular sinus; SIGN-R1, specific intracellular adhesion molecule-grabbing nonintegrin R1; SSM, subcapsular sinus-lining
macrophage.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
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Since the original proposal by Fearon and Locksley
(Fearon and Locksley. 1996. Science 272: 50–53) that
the complement system linked innate and adaptive
immunity, there has been a rapid expansion of studies
on this topic. With the advance of intravital imaging, a
number of recent papers revealed an additional novel
pathway in which complement C3 and its receptors
enhance humoral immunity through delivery of Ag to
the B cell compartment. In this review, we discuss this
pathway and highlight several novel exceptions recently
found with a model influenza vaccine, such as mannosebinding lectin opsonization of influenza and uptake by
macrophages, and the capture of virus by dendritic cells
residing in the medullary compartment of peripheral
lymph nodes. The Journal of Immunology, 2010, 185:
2659–2664.
2660
BRIEF REVIEWS: COMPLEMENT IN ADAPTIVE IMMUNITY
FIGURE 1. The conduit network, formed by collagen
fibers, is secreted by the FRCs and drains small Ags from
the SCS area of the LN to the B cell follicle. FDCs present
in the follicle are closely associated with the collagen fibers.
Right panel shows an electron micrograph of the SCS
area. The arrow indicates a conduit opening into the sinus
(S.F. Gonzalez, unpublished results). Scale bar, 2 mm.
MF, macrophage; SCS, subcapsular sinus.
phages (MMs), which are more similar to marginal zone
(MZ) macrophages of the spleen, express specific intracellular
adhesion molecule-grabbing nonintegrin R1 (SIGN-R1) and
mannose receptor (MR), in addition to CR3. However, SSMs
are more similar to the metallophilic macrophages of the MZ
and express metallophilic macrophages Ab-1 (17).
B cell transport of C3-ICs is CD21/35 dependent
In mice, complement receptors CD21 and CD35 are encoded
at the Cr2 locus (19, 20). CD21 represents a splice product of
the CD35 mRNA, whereas in humans they are encoded at
separate loci (21, 22). Murine CD21 and CD35 (CD21/35)
are coexpressed on B cells, FDCs, and a subset of T cells.
CD35 binds activated C3b and C4b and, like CD21, it binds
the cleavage products iC3b, C3d,g, and C3d covalently
FIGURE 2. A, Pathways for the circulation of Ag in the LN. 1, Complement C3 opsonizes Ag in the presence of
Ab. C3-ICs are formed by the deposition of complement proteins and IgG
on the surface of the Ag. 2, The retention of C3-ICs on the surface of the
SSMs is CR3 and FcRIIb dependent.
3, B cells transport C3-ICs from the surface of the SSMs to the FDCs in a
CD21-dependent manner. 4, C3-ICs are
transferred to FDCs. 5a, Cognate B cells
acquire small Ag drained via FRC conduits directly or (5b) from the FDC surface. B, Influenza virus uptake by SSMs
is MBL dependent.
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not go through a lysosomal compartment (18). Therefore, it is
possible that C3-ICs are partially protected by this cycling process. It is not clear how C3-ICs are actually transferred to B cells;
however, uptake on the naive B cells is CD21/35 dependent
and highly efficient. Strikingly, .25% of naive B cells within
the LN follicles take up C3-ICs within 8 h of s.c. injection of
Ag into immune mice (9, 16).
In naive mice in which Ab does not pre-exist, it is not clear
how large protein Ags within the lymph are captured by sinuslining macrophages. The lymph, in general, is thought to
include a similar repertoire of recognition proteins that activate
complement as found in blood, such as mannose-binding lectins (MBL), ficolins, and pentraxin. Alternatively, protein Ags
might be directly taken up by scavenger or lectin receptors expressed on the sinus-lining macrophages. Medullary macro-
The Journal of Immunology
FIGURE 3. Complement receptors
CD21 and CD35 play an important
role in at least three stages of B cell
differentiation. Stage 1: Coligation of
C3d-Ag with BCR lowers threshold of
B cell activation, leading to migration
of the activated B cell to the T cell–
B cell boundary, where cognate interaction occurs and B cells receive costimulation via CD40. Stage 2: Activated B cells enter a GC where they
begin further differentiation, including
rapid cell division, somatic cell hypermutation, and class switch recombination. Stage 3: Following clonal selection
(binding of Ag on FDC), the GC B cell
differentiates into an effector cell (plasma cell) or memory B cell. Maintenance
of B effector and memory cells is dependent on presence of Ag on FDC.
discussed above, naive B cells acquire C3-ICs from SSMs lining
the SCS via their CD21/35 receptors. Whether uptake induces
a direct signal via CD21/35 or involves the coreceptor CD19
and CD81 is not clear. In a recent study using mice in which
CD21/35 are uncoupled from CD19 (Cr2D/D), naive B cells
in the mutant line seemed to take up C3-ICs normally and
deliver them to FDCs (42). CD21/35 receptors include a cytoplasmic domain so it is possible that uptake of C3-ICs induces
a signal by CD21, independent of CD19 (52), and triggers
B cell migration to the FDCs. An alternative possibility is
that B cells within the follicles migrate constitutively to FDCs
based on a chemokine gradient in a manner similar to that
described for MZ B cells (53); therefore, direct signaling by
C3-ICs may not be essential. Thus, as B cells enter the peripheral LNs via high endothelial cells, they cycle to the SCS
area, which is rich in marginal reticular cells that secrete
B cell chemokines (54), and subsequently to FDCs, which
are also a source of chemokines (45).
Earlier studies identified a role for C3 and CD21/35 in the
capture of ICs by MZ B cells and transport to FDCs within
B cell follicles (55–57). Using intravital imaging, Cyster and
colleagues (53) identified a similar pathway for capture of
C3-ICs in the spleen as reported for the LNs. They found that
MZ B cells continuously shuttled between the splenic MZ
and the B cell follicles by a CXCR5-chemokine-sphingosine
1-phosphate receptor-dependent mechanism. Thus, naive
MZ B cells capture C3-coated Ags within the MZ sinus
and transport them into the follicles where they are transferred to FDCs. How C3-ICs are handed off to FDCs is
not clear; however, uptake on the FDCs is dependent on
CD21/35 and FcRIIb. Because the latter receptors (FcRIIb)
are not constitutively expressed but are upregulated following
FDC activation, complement receptors are critical for the
initial capture and retention of C3-ICs (40).
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attached to Ags (19, 23–25). They play a critical role in
humoral immunity because blocking with Abs (26, 27), a
soluble receptor (28), or deletion of the receptors (29, 30)
leads to impaired humoral immunity to model protein Ags,
bacteria (31), and viruses (32, 33) (Fig. 3). On B cells,
CD21/35 forms a coreceptor with CD19 and CD81, and
coligation with the BCR lowers the threshold of B cell activation (34–36).
A second major function of CD21/35 is retention of Ag on
FDCs. They represent the major Ag receptors on FDCs (37–
39). Ag is also retained for long periods via FcRIIb; however,
its expression is not constitutive but upregulated on activated
FDCs (40). FDCs are thought to be the major source of Ag
required for clonal selection of B cells during the germinal
center (GC) reaction (41) and possibly for maintenance of
memory (42–44). They retain Ag and secrete B cell growth
factors and the B cell attractant CXCL-13, as mentioned
above (45). Mice deficient in C3 or CD21/35 have fewer
and smaller GCs, and B cells fail to survive (46, 47).
There is growing evidence that B cells acquire Ag and are
activated most efficiently when it is attached to membrane (48–
50). Therefore, retention of Ag and C3d on the FDC surface
could enhance formation of the B cell synapse. Support for a
role for FDCs as an important source of Ag comes from the
recent studies of Cyster et al. (51); they demonstrated direct
capture of Ag from FDC surface by cognate B cells using
multiphoton intravital microscopy (MP-IVM). Importantly,
they also identified a role for CD21/35 in efficient uptake of
medium-affinity Ags. In their model system, MD4 B cells deficient in CD21/35 acquired less Ag from FDCs. One explanation for their results is that the coreceptor acts to enhance
BCR signaling in the synapse between the BCR and FDC.
A third novel role for CD21/35 is transport of lymph-borne
C3-ICs into the B cell follicles and transfer to FDCs. As
2661
2662
Influenza virus is captured via a novel pathway
Medullary dendritic cells bind influenza
In addition to capture of virus by sinus-lining macrophages,
Gonzalez et al. (63) reported that a resident population of
FIGURE 4. Lymph-borne UV-inactive influenza (strain
PR8) fills the SCS of the LN, where it is partially taken
up by SSMs. Binding of virus to SSM is MBL dependent.
Virions are channeled to the LN medulla, where MMs and
resident DCs capture them in a SIGN-R1–dependent
manner. Model speculates that C1q is activated by SIGNR1, leading to C3 activation and deposition on PR8. Subsequently, resident DCs transport C3-coated virus complex
to the follicles, where it is transferred to FDCs.
dendritic cells (DCs) bound virus in the medullary region. Using
flow-cytometric assays, they identified a CD11c+ CD11bhi,
SIGN-R1+ population that bound a significantly large amount
of virus. Interestingly, ∼50% of the PR8-binding DCs expressed
a receptor for the cysteine-rich (CR) domain of the MR based
on binding with an Ig Fc fusion protein with the CR domain
(i.e., CR-Fc) (66). The receptor for the CR domain of MR is
associated with dendritic-like cells that accumulate within the
B cell follicles following immunization and possibly represent
cells transporting Ag (67).
Studies by Park and colleagues (68) identified murine SIGNR1 as the major receptor in the uptake of Streptococcus pneumoniae by MZ macrophages. SIGN-R1 is structurally similar to
human DC-SIGN and is a C-type lectin that binds glycans rich
in mannose, such as dextran and capsular polysaccharides of
pneumococcus (69, 70). It is the major receptor for S. pneumoniae, because pretreatment of mice with a monoclonal Ab
(clone 22D1) that downregulates macrophage expression of
SIGN-R1 led to an impaired humoral response and increased
mortality (68). Notably, binding of bacteria or purified polysaccharide by SIGN-R1 induces receptor aggregation, activation of C1q, and C3 deposition (68). Mice deficient in C3
have impaired immunity to S. pneumoniae, and this is associated with impaired uptake of bacteria on FDCs. Thus, one
interpretation of the findings is that opsonization of bacteria
with C3 via SIGN-R1 is critical for uptake on FDCs and access
by follicular B cells.
SIGN-R1 is required for local humoral immunity to influenza
Using a similar approach as Park and colleagues (68), Gonzalez
et al. (63) showed that treatment of mice with a mAb to
SIGN-R1 that downregulates receptor expression significantly
reduced the uptake of UV-PR8 by resident DCs. Moreover,
blocking of UV-viral uptake via SIGN-R1 in MBL2/2 mice
substantially reduced the capture of UV-PR8 in the draining
LN, and the local B cell response was reduced by 3-fold.
However, in these experiments, the systemic response was
not affected, suggesting that blocking of viral capture locally
led to efficient spreading of the virus and response in downstream LNs and spleen. Further support for the importance of
the resident DCs in viral capture and humoral immunity
was determined by the elimination of DCs systemically in
CD11c-DTR bone marrow chimeric mice (71). In this model,
CD11chi cells express the monkey diphtheria toxin receptor and
are sensitive to ablation on treatment with diphtheria toxin.
Thus, the CD11c DC-ablated chimeric mice had a significantly
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Influenza infection represents a major pathogen of humans; in
general it is controlled by annual vaccination and induction of
a protective humoral response (58–60). Complement C3 and
CD21/35 are important components in humoral immunity to
influenza, based on earlier studies (33, 61, 62). Although much
of the focus on the enhancing role of complement has been on
the B cell coreceptor, the C3-CD21/35 pathway is also important
for transport and retention of Ag on FDCs, as discussed above.
Recent studies identified a novel pathway by which viral Ag is
captured and possibly transported to the FDCs. Gonzalez et al.
(63) used a UV-inactivated form of influenza virus (strain PR8)
as a model vaccine. Using a fluorescent-tagged form of UVPR8 injected in the footpad (s.c.) and imaging by MP-IVM,
they identified rapid filling of the sinus and capture by SSMs
and MMs. As discussed earlier, macrophages lining these
regions represent distinct populations, as distinguished by
functional maturity and cell surface markers (17).
The study by Gonzalez et al. (63) reported that labeled
UV-PR8 virus was rapidly bound by the SSMs and MMs,
similar to that reported for vesicular stomatitis virus (13). In
contrast to the earlier studies, UV-PR8 was not retained on the
surface but was internalized. Binding and phagocytosis was
dependent on MBL, because SSMs in MBL2/2 mice failed
to bind the virus. Opsonization by MBL is in agreement with
earlier studies by Ezekowitz and colleagues (64), who reported
that guinea pig mannose-binding lectin bound and neutralized
influenza. Interestingly, blocking of UV-PR8 uptake by SSMs
alone or SSMs and MMs did not impair humoral immunity.
This was somewhat surprising because earlier reports suggested
that capture of particulate Ags by SSMs was important for
activation of cognate B cells. However, capture of UV-PR8
by sinus-lining macrophages was important in limiting the
spread of the virus to downstream LNs and the spleen. Thus,
the role of macrophages lining the sinus of peripheral LNs is
important in the efficient capture and spread of virus, but it is
not important for humoral immunity to inactive influenza.
It is not clear how MBL-opsonized UV-PR8 is phagocytosed by
SSMs. Because a known receptor for murine MBL is not clear it
is possible that uptake is via a complement ligand, because the
lectin pathway activates C2 and C4 or C3 directly via a bypass
pathway, as described in human serum (Fig. 2B) (65).
BRIEF REVIEWS: COMPLEMENT IN ADAPTIVE IMMUNITY
The Journal of Immunology
Conclusions
In this review, we discussed a novel role for complement in the
transport of soluble Ags, ICs, and viral Ags to the B cell compartment. The finding that complement C3 and its receptors
(i.e., CD21/35 and CR3 [CD11b/18]) participate in the capture
and transport of lymph-borne Ags to the FDCs provides another
important mechanism by which complement enhances adaptive
immunity. Moreover, the identification of a network of conduits that directly funnel small Ags to the FDCs further elucidates how Ags are made accessible to naive B cells. We also highlight a new pathway in which capture of inactive influenza via
the SIGN-R1 receptor signals the resident DCs to migrate toward the B cell compartment. It will be important, in future experiments, to determine whether C3 is activated and deposited
on the virus following binding by SIGN-R1 and to track resident
DCs with bound PR8 into the B cell follicles and determine
whether they hand-off viral Ag directly to FDCs (Fig. 4).
Acknowledgments
We thank Alex Gillmore for assistance with assembly of the manuscript and
figures and members of the laboratory for discussions on Ag trafficking.
Disclosures
The authors have no financial conflicts of interest.
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