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Chapter 24 Complement and Immunoglobulin Biology Leading to Clinical Translation 269

mice producing only membrane IgM (i.e., with gene-targeted dele- structures and biochemical data, the nature of this important interface
tion of secretory signals) produce significantly reduced antibody had been hotly debated for a decade because of discrepancies between
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responses to T-dependent antigens. A second mechanism for initial a structure of the CR2(SCR1-2):C3d complex published in 2001
CP activation on the antigen, which may be relevant to a subset of with both preexisting and subsequent biochemical data in the litera-
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antigens bearing a repeating epitope, involves binding of the antigen ture. A 2011 de novo structure of this complex, depicted in Fig.
by B cells through the surface IgM of two or more B-cell receptors 24.4C, appears to have resolved the issue because the interactions
(BCRs). The cross-linking and distortion that is imparted to the Fc µ seen in the new structure are fully supported by the biochemical data
regions of adjacent BCRs is sufficient to activate the CP at the B-cell in the literature. For example, the biochemical data suggesting that
surface. Indeed, this mechanism does not work if the BCR µ-chain there should be multiple ionic bonds mediating the binding is fully
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contains a mutation that abolishes C1q binding. Finally, a third rationalized in terms of the five such bonds seen in the structure
permutation of these mechanisms may apply to monovalent soluble between a very negatively charged interface on a concave face of C3d
T-dependent antigens. The antigen is first captured by the BCR, that is remote from the covalent attachment site and positively
creating an antigen array on the B-cell surface, to which low-affinity charged lysine and arginine side chains from CR2 sticking down and
natural repertoire IgM can bind by virtue of avidity effects and initi- interacting with oppositely charged residues on the C3d interface, as
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ate the CP. As illustrated by these examples, immune complex can be appreciated in Fig. 24.4C.
formation is only important for initiating the CP, leading to the As a coreceptor, engagement of CD21 by complement-coupled
deposition of C3 activation products on the antigen or immune antigen on the surface of a B lymphocyte, in combination with
complex. Indeed, antigens directly conjugated to C3b or C3d frag- membrane Ig (BCR) cross-linking, would lower the threshold of
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ments are more potent immunogens compared to unconjugated signal through the BCR required to activate the cell. Accordingly,
antigen. 92,93 Furthermore, the magnitude of the immune response is naive B lymphocytes bear low-affinity receptors for antigen; therefore,
directly influenced by the number of C3d fragments conjugated especially under conditions of limiting antigen, as would be the case
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to the antigen. Therefore activated products of complement com- during initial encounter with a microbial pathogen, additional signal-
ponent C3 act as a natural adjuvant in driving efficient antibody ing by the CD21 coreceptor is required for efficient activation. This
responses. was demonstrated in vitro by culturing B lymphocytes with cognate
antigen, either uncoupled or coupled to C3d. By measuring intracel-
2+
Complement Receptors and Antibody Responses lular Ca levels as a measure of cell activation, it was estimated that
100- to 1000-fold less C3d-conjugated antigen was required to
activate B lymphocytes compared with unconjugated antigen. 92
B-Lymphocyte Coreceptors The opportunity to test the importance of CD21 and CD35 as
B-lymphocyte coreceptors in vivo came from studies using mice with
The effects of complement-coated antigens on antibody responses are targeted disruption in the Cr2 locus. Importantly, Cr2-deficient mice
mediated primarily through complement receptors CD21 and CD35. have impaired humoral responses similar to C1q-, C4-, and
CD21 and CD35 are expressed predominantly on B lymphocytes C3-deficient mice (Fig. 24.5). 109–111 Using embryonic stem cells with
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and FDCs. 94,95 CD35 is also found on polymorphonuclear cells, a disrupted Cr2 locus, Croix et al used blastocyst complementation
−/−
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macrophages, mast cells, and DCs. CD21 and CD35 are encoded of Rag2 mice, such that chimeric mice expressed CD21/CD35 on
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for by separate yet closely linked genes in humans. In mice, CD21 FDCs but not on B lymphocytes. These chimeric mice displayed
and CD35 originate from the same locus (Cr2) and are generated by impaired antibody responses to the T-dependent antigen NP-KLH
alternative splicing events at the RNA level. 97,98 compared with control subjects. Therefore CD21/CD35 on B lym-
Two novel sets of experiments demonstrated that CD21 and phocytes is important for normal antibody responses. Although
CD35 are important in regulating B-lymphocyte responses to CD21/CD35 on FDC is on its own, insufficient for normal antibody
T-dependent antigens. In the first set of experiments, antibodies responses, as discussed in the next section, CD21/CD35 on FDC
specific for both CD21 and CD35 or CD35 alone were administered does have a specific role in the memory response of B cell–mediated
to immunized mice. 99–102 In the second set of experiments, a soluble immunity.
form of CD21 was administered to immunized mice, thereby com- The covalent attachment of complement to antigen engages
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peting for C3d-coupled antigen interactions. In both sets of CD21 as a complex with CD19/CD81 and BCR on the cell surface
experiments, treatment impaired antibody responses. In the first (see Fig. 24.4A). 104,113,114 Dual binding of CD21/CD19/CD81 with
approach, the antibody that specifically blocked the interaction of BCR generates a stronger signal compared with BCR engagement
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C3d with CD21 was much more effective at blocking antibody alone. If the combined signal is sufficient, the B lymphocyte is
responses compared with anti-CD35 antibody treatment, which activated. If insufficient, then the B lymphocyte is likely eliminated
blocked only the binding of C3b to CD35. This suggested that by apoptosis. 87,115–119 The major ligand-binding receptor within the
although both receptors contribute, CD21 is more important in CD21/CD19/CD81 complex is CD21. The major role of CD19 is
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regulating antibody responses. 100 in initiating a signaling cascade within the cell. CD81 is a tetra-
Because CD21 and CD35 are found on B lymphocytes and spanning molecule that stabilizes the complex within the membrane.
FDCs, two important cell types for humoral responses, two nonmu- After coligation of the BCR with the CD21/CD19/CD81 complex,
tually exclusive models are proposed for their function. In the first CD81 gets S-palmitoylated on a cysteine side chain, and this in turn
model, CD21 augments antibody responses through activity as a mobilizes the coligated complexes to a special compartment of the
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coreceptor on B lymphocytes (Fig. 24.4A). The second model plasma membrane known as a lipid raft. Localization to this compart-
proposes that CD21/CD35 on FDCs trap and focus antigen such ment facilitates prolonged intracellular signaling because the com-
that B lymphocytes can efficiently cross-link their antigen receptor partment is rich in signal-propagating phosphokinases but is relatively
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to become activated (Fig. 24.4B). devoid of the regulatory phosphatases. Absence of any of the
As is apparent from the schematics in Fig. 24.4A–B and as will CD21/CD19/CD81 components adversely affects antibody responses
be elaborated upon further in the ensuing discussion, the key ligand to T-dependent antigens, although the degree of impairment
receptor–receptor interaction mediating the linkage between comple- varies. 109,122–124
ment and the adaptive humoral immune system is that between the
C3d fragment that is covalently coupled to antigen and CD21 (CR2)
present on B cells and FDC. The extracellular region of CD21 is Focusing Antigen on Follicular Dendritic Cells
composed of 15 or 16 SCR domains (because of the usage of alterna-
tive splice sites for exon 11), but the C3d binding site is confined to The second role of complement receptors CD21 and CD35 in regu-
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the two N-terminal–most SCR domains. In what is an instructive lating humoral responses is that they permit FDCs to trap antigen
lesson on the need to have concordance between x-ray crystallographic (Fig. 24.6). 105,125 FDCs concentrate in regions of ongoing immune