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KEY WORDS: complement receptors, B cell coreceptor, clonal selection, innate immunity, B-1 lymphocytes, germinal centers, follicular dendritic cells
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ABSTRACT |
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Covalent attachment of activated complement C3 (C3d) to antigen links innate and adaptive immunity by targeting antigen to follicular dendritic cells (FDC) and B cells via specific receptors CD21 and CD35. Recent characterization of knockout mice deficient in complement components C3, C4, or the receptors CD21 and CD35 as well as biochemical studies of the CD21/CD19/Tapa-1 coreceptor on B cells have helped to elucidate the mechanism of complement regulation of both B-1 and B-2 lymphocytes. Interestingly, natural antibody of the adaptive immune system provides a major recognition role in activation of the complement system, which in turn enhances activation of antigen-specific B cells. Enhancement of the primary and secondary immune response to T-dependent antigens is mediated by coligation of the coreceptor and the B cell antigen receptor, which dramatically increases follicular retention and B cell survival within the germinal center. Most recent evidence suggests that complement also regulates elimination of self-reactive B cells, as breeding of mice that are deficient in C4 or CD21/CD35 with the lupus-prone strain of lpr mice demonstrates an exacerbation of disease due to an increase in autoantibodies.
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INTRODUCTION |
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Participation of the complement system in regulation of B cell responses is a relatively old idea that was first proposed based on the finding of complement receptors on murine lymphocytes by Lay and Nussenzweig (1, 2). Subsequently, Pepys found that transient depletion of the third component (C3) resulted in an impaired humoral response to T-dependent and T-independent antigens (3). Complement was found to be essential for localization of immune aggregates to the follicular region of the secondary lymphoid compartment, and this suggested a mechanism for its enhancing effect (4, 5). Studies with guinea pigs deficient in C3 or components involved in its activation such as C2 and C4 confirmed the importance of complement in formation of a primary and secondary immune response to protein antigens (reviewed in 6). However, the absence of well-defined reagents for dissecting the humoral response limited more detailed analyses in these animal models.
In mice, follicular dendritic cells (FDC) and B cells bear two distinct receptors, i.e. CD21 (l50 kDa) (7, 8, 9) and CD35 (l90 kDa) (10) for activated products of C3 and C4 (Figure 1). They are encoded at a single locus (Cr2) on chromosome 1 (8). The two receptors are assembled from multiple repeating structures referred to as short consensus repeats (SCRS) that consist of conserved units of 60–70 amino acids. CD21 is assembled from 15 SCRS, a transmembrane region, and a 35 amino acid cytoplasmic tail, whereas CD35 includes all of CD21 and an additional six SCRs on its N-terminal region. The two receptors differ in their ligand-binding specificity as CD21 binds iC3b, C3d,g, C3d, and C4d; and CD35 includes additional binding sites for C3b and C4b in the N-terminal region (11, 12, 13). CD35 has an additional function as a cofactor to factor I in the conversion of C3b to iC3b and C3d,g (10, 13).
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Human FDC and B cells also coexpress CD21 and CD35; however, the two genes are encoded at separate loci (14, 15) and appear to have distinct functions. Biochemical characterization of human complement receptor CD21 led to the novel finding that CD21 forms either a complex with CD35 (16) or CD19, TAPA-1, and Leu-13 on B lymphocytes (17, 18, 19). Coligation of the CD21/CD19 receptor complex with the B cell receptor (BCR) enhances signal transduction and effectively reduces the amount of antigen required by 10–100-fold (20, 21). Thus, CD21/CD19 participates as a coreceptor on human B cells much as does CD4 on T cells (22) and so effectively lowers the threshold for BCR signal transduction (23, 24). Since a ligand for CD19 has not been reported, it is assumed that CD21 binding of its ligands, i.e. iC3b, C3d,g, C3d, provides the primary recognition site for the coreceptor. This finding led to an alternative hypothesis to explain the enhancing role of C3 in humoral immunity. The involvement of CD21 in the humoral response to T-dependent antigens was confirmed in vivo by the demonstration that pretreatment of mice with either a CD21/CD35 specific-monoclonal antibody (mab) (25, 26) or a fusion protein of CD21-IgG (sCR2) (27) blocked the secondary humoral response. However, because both FDC and B cells express CD21, these studies could not distinguish between the two proposed hypotheses. Although not mutually exclusive, whether complement enhances humoral immunity by lowering the threshold of B cell signal transduction via the coreceptor or by increasing the efficiency of antigen trapping and retention on FDC via CD21/CD35 (Figure 2) remains a question.
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In this review, I discuss recent studies that address the mechanism for complement regulation of B cell development within the secondary lymphoid compartment. Furthermore, I attempt to combine results from studies of complement in both inflammation and the humoral response. This rather broad approach has led to our current model that complement activated principally by natural antibody modulates the selection and specific response of both B-1 and B-2 lymphocytes via the CD21/CD19 coreceptor. Thus, innate immunity regulates adaptive immune responses and natural antibody, which is the product of B-1 cells and is important in activation of the classical complement pathway. Finally, I speculate that complement is an important factor in setting the threshold for negative selection of B cells.
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HOW IS COMPLEMENT ACTIVATED IN THE IMMUNE RESPONSE? |
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Models explaining an enhancing role for complement are based on the assumption that C3 becomes activated and binds covalently to protein antigens in vivo as it does in vitro (28). Thus, in order either to coligate CD21/CD19 coreceptor and BCR or to trap antigen on FDC, the CD21 ligand C3d must first become attached to antigen. Definitive evidence that direct attachment of C3d to antigen results in an enhanced humoral response was demonstrated by Dempsey et al (29). They found that fusion proteins of C3d and lysozyme were highly immunogenic. Thus, coupling two and three copies of C3d to the N-terminus of lysozyme reduced the amount of antigen required to induce an optimal secondary antibody response by two and three orders of magnitude, respectively. Given the importance of C3 binding, it is worth considering how C3 is activated following immunization or infection and becomes attached to antigen in vivo.
Recognition Molecules of Innate Immunity
The innate immune system has evolved highly specific recognition
proteins that activate complement following binding of their
ligand (30) such as mannan binding lectin (MBL) (31, 32),
C-reactive protein (33), complement system (34, 35), and natural
antibody. For example, MBL has evolved highly specific structures
(carbohydrate recognition domains) that bind carbohydrates common
to pathogens. The classical pathway of the complement system can
be activated directly by the binding of C1q to pathogens such as
human immune deficiency virus (36). In the alternative pathway of
complement, the spontaneous activation of C3b and covalent
attachment to non-self surfaces, which are not protected by
sialic acid or regulators of complement, lead to amplification of
C3 activation and attachment of C3b (37).
Natural Antibody Is a Major Recognition Molecule of Innate
Immunity
Serum immuoglobulin is one of the most abundant proteins in the
blood (approximately 10 mg/ml). Natural antibodies account for a
large part of circulating Ig; however, despite their abundance,
their functional importance remains controversial. The primary
source of natural antibody in the mouse is the peritoneal B-1
cell (38). This CD5+ subset of B cells is
self-replenishing and is not thought to produce hypermutated
antibodies. The repertoire of B-1 cells appears to be biased
toward germline-encoded antibodies that react with structures
common to pathogens, such as levan, LPS, and phosphatidyl choline
(PtC), with a wide range of binding affinities (39, 40, 41). For
example, 10–20% of the B-1 cell repertoire recognizes PtC, LPS,
and epitopes on bromelain-treated red blood cells (42, 43). The
heavy and light chain genes represent germline products of two
heavy and light chain V region subfamilies, i.e. VH11Vk9 and
VH12Vk4 (39, 42). CD5+B cells also are a source of
polyreactive IgM natural antibodies, which react with
self-antigens (44). Natural antibody also binds to exogenous
antigens such as keyhole limpet hemocyanin (KLH) and activates
complement (45). Given the specificity of many of the products of
B-1 cells, it is probable that the repertoire evolved to provide
immediate protection against pathogens. As such, natural antibody
is an important recognition molecule of innate immunity.
Mice deficient in immunoglobulin are highly sensitive to bacterial infection. Reconstitution of Ig-deficient mice (X-linked immunodeficiency, or Xid) with the PtC-specific IgM is protective against infection with streptococcus (46). Protection is probably mediated via the classical pathway as mice deficient in C4 also have an increased sensitivity to infection with group B streptococcus (47). The recent finding that mice deficient in classical pathway complement were more susceptible to endotoxemia than WT controls (48) led to the observation that natural antibody is essential in intravascular clearance and protection against high doses of endotoxin (49). The source of the antibody was identified to be peritoneal B-1 cells as mice with a disruption of the gene encoding Bruton's tyrosine kinase (Btk-/-), the enzyme deficiency that results in Xid, are nearly as sensitive to endotoxin shock as those totally deficient in Ig, i.e. recombinase activation gene 2 deficient (RAG-2-/-) (49). Btk-deficient mice express negligible levels of IgM and IgG3 but a normal range of the other subclasses of IgG (50). IgM is the major protective isotype, as reconstitution of the IgM/IgG3 deficient mice with pooled IgM is protective against endotoxin shock. In vitro binding experiments confirmed that the IgM fraction of pooled serum rather than IgG contained natural antibody that bound the LPS moiety expressed by Salmonella (49).
Natural antibody not only recognizes pathogenic organisms but can recognize self-antigens and initiate autoimmune injury. One such example comes from recent studies on ischemia reperfusion injury. Reperfusion of hypoxic tissue following occlusion of blood supply results in a local inflammatory response leading to irreversible injury of both endothelium and skeletal muscle. Injury is mediated in part by the complement system as pretreatment of animals with a soluble form of the CR1 receptor (which inactivates C3 convertase) reduces injury substantially (51, 52). The first evidence that reperfusion injury was initiated by natural antibody came from the two findings: that inflammation was mediated by the classical pathway of complement and that deposits of IgM and C3 colocalized at the site of inflammation following reperfusion (53). This hypothesis was confirmed with antibody-deficient mice (RAG-2-/-) that were protected against injury; this protection was abrogated by reconstitution with either fresh mouse serum (53) or pooled IgM (54). A model that would explain these observations is that neoepitopes are expressed on hypoxic venular endothelium and that circulating natural antibody recognize the altered surface and bind. This results in activation of the classical pathway of complement and initiation of an inflammatory response. These findings are relevant to this review as they provide an insight into the antibody repertoire of B-1 lymphocytes, discussed in the next section.
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ROLE OF COMPLEMENT IN CLONAL SELECTION OF B-1 CELLS |
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One of the phenotypes identified in mice bearing a targeted disruption of the Cr2 locus (Cr2-/-) is a selective reduction in the B-1a (CD5+, IgM+, CD11b/CD18+) subset of peritoneal B-1 cells (55). Thus, while the B-1b (IgM+, CD11b/CD18+) subset of B-1 cells appears normal, the B-la population is reduced by approximately 50%. Despite this reduction in cell number, the level of IgM in the serum of Cr2-/- mice is normal. Interestingly, mice deficient in CD19 (CD19-/-) have an even greater reduction in B-1 cells with a corresponding decrease in serum IgM (56, 57). The more severe phenotype of the CD19-deficient mice could reflect an inherent role for CD19 signaling independent of CD21.
B-1 cells represent a self-replenishing population of B lymphocytes that are thought to be maintained or expanded by contact with antigen such as enteric bacteria. The finding that mice deficient in the CD21/CD19 coreceptor have a reduced number of peritoneal B-1 cells suggests an essential role for complement in selection of this population of lymphocytes. Thus, contact of B-1 cells with C3d-coated antigen might enhance cell signaling and clonal selection via coligation of the coreceptor with BCR, in a manner similar to that proposed for enhancement of B-2 lymphocyte signaling (24; Figure 2). A prediction of this hypothesis would be that the repertoire of natural antibody in Cr2-/- mice is reduced or altered compared to WT mice. To test this hypothesis, Cr2-/- mice were examined in the natural antibody-dependent ischemia reperfusion model (53, 54). Interestingly, they are equally protected in the mucosal ischemia reperfusion model as mice deficient in either C4 (C4-/-) or Ig (RAG-2-/-) (A Prodeus, RR Reid, H Hechtman, FD Moore, MC Carroll unpublished results). Protection is due to an absence of specific IgM, as reconstitution of Cr2-/- mice with pooled IgM abrogates protection. Thus, even though the CD21/CD35-deficient mice have normal circulating levels of IgM, they lack the natural antibody/antibodies that mediate reperfusion injury; and consequently they are protected in the IgM-dependent reperfusion model. The source of specific IgM is the B-1 lymphocyte because engraftment of Cr2-/- mice with peritoneal B-1 cells harvested from WT mice restores normal inflammation in the mucosal reperfusion injury model (A Prodeus, RR Reid, H Hechtman, FD Moore, MC Carroll, unpublished results). The results support the hypothesis that B-la cells require antigen stimulation for survival and expansion and that selection is CD21 dependent (Figure 3).
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Source of C3
The complement system consists of over 20 serum proteins that
interact in a series of proteolytic events leading to release of
proinflammatory peptides (C3a and C5a), covalent attachment of
opsonins (C3b, iC3b, C3d, C4b, C4d) to surfaces and formation of
the membrane attack complex (C5b-9) (34, 35). C3 protein is a
major component of blood and is found at a concentration of
approximately 1 mg/ml. While the primary site of synthesis is the
liver (58), extra-hepatic synthesis is common and a number of
cell types such as macrophages (59), keratinocytes (60), kidney
tubular epithelial cells (61), and endothelial cells (62)
synthesize C3 protein. Synthesis is regulated by inflammatory
cytokines such as IL-l
(61), IL-6 (63), and
interferon-gamma (IFN-
) (64) that are known to upregulate
acute phase proteins.
Results from our early studies with mice bearing heterozygous deficiency in C3 demonstrated a gene dosage effect as the mice responded to T-dependent antigens with a response intermediate between that of wild-type controls (WT) and animals totally deficient in C3 (C3-/-) (MB Fischer, M Ma, MC Carroll, unpublished results). This was something of a surprise given the relatively high level of C3 in blood, and it suggested that its concentration within the tissues may be a limiting factor in enhancement of humoral immunity. To test the importance of locally synthesized C3 in the humoral response, C3-deficient mice that have an impaired response to T-dependent antigens (discussed in more detail below) (65) were reconstituted with bone marrow (BM) from WT mice. Interestingly, engraftment of the deficient mice with WT BM rescued their impaired humoral response to (4-hydroxy-3-nitrophenyl) acetyl keyhole limet hemocyanin (NP-KLH) despite nearly undetectable levels of C3 in their blood (66). In situ hybridization and immunohistochemical analyses of cryosections of the spleens of immunized WT mice and BM chimeras, localized C3 synthesis to the white pulp region. The cellular source of C3 protein and mRNA was further identified as MOMA-2+ macrophages by immunohistochemical staining and by reverse-transcriptase-PCR analysis of RNA isolated from purified MOMA-2+ cells, respectively (66). This population of macrophage is located primarily within the T and B cell zones of the secondary lymphoid compartment (67). Thus, in the case of the BM chimeras, a sufficient level of C3 protein is synthesized within the lymphoid compartment by WT donor macrophages to provide enhancement of the humoral response and efficient trapping of antigen on FDC.
Local synthesis appears to be tightly regulated as C3 mRNA
expression is not detectable in the lymphoid compartment in
nonimmune WT mice. However, within 24 h following immunization
C3mRNA is expressed by MOMA2+ macrophages within the
spleen and lymph nodes and persists for 3 to 5 days or up to 2
weeks following primary and secondary immunizations, respectively
(66). While the molecular events regulating C3 expression have
not been identified, it seems most likely that inflammatory
cytokines such as IL-la, IL-6, and IFN- as discussed above
are involved (Figure 4).
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REGULATION OF ADAPTIVE IMMUNITY BY CD21/CD35 |
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A distinct role for classical pathway complement in enhancement
of the humoral response was confirmed in mice genetically
deficient in C3 or C4 (65). Comparison of the primary and
secondary immune response of these mice with WT mice following
challenge with T-dependent antigen (bacteriophage (
X 174)
demonstrated that while their T-cell response was normal, their B
cell response was impaired (65) (Figure 5).
Phage antigen was
selected as a model T-dependent antigen because of the sensitive
plaque forming unit (PFU) assay for detecting antibody and it was
used in previous studies in guinea pigs deficient in C3 or C4
(68, 69). As expected, splenic B cells of C3 and C4 deficient
mice respond normally in proliferation assays in vitro when
stimulated via their BCR (crosslinking IgM receptor) or CD40.
Likewise, no difference in the response to LPS was observed among
B cells isolated from WT or the deficient mice. It was not
unexpected that B cells in C3 deficient mice would respond
normally in vitro as these assays did not involve complement
ligands.
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The immune-enhancing role of complement has been proposed as mediated by the CD21 receptor based on blocking experiments [anti-CD21 antibody (26) and sCR2 (27)] and biochemical studies (23, 24) as discussed above. The recent availability of mice deficient in CD21 and CD35 (Cr2-/-) has provided a mouse model not only for confirming the critical role for this receptor/receptors in humoral immunity but for dissection of the mechanism. As predicted, the Cr2-/- mice have an impaired humoral response to T-dependent antigens (T-D) characterized by a reduction in the secondary antibody (55, 70). In general, the phenotype of the Cr2-/- mice is very similar to that of mice deficient in C3 or C4 because they have a reduced number and size of germinal centers and a corresponding reduction in serum levels of the switched isotypes of Ig, i.e. IgG2a, IgG2b, and IgG3 (55). Interestingly, some GC do form in response to immunization with soluble T-D antigen, but their mean size is approximately 10-fold less than that found with WT mice; and this is discussed in more detail below. In the absence of complement in in vitro assays, splenic B cells isolated from Cr2-/- mice appear to transduce signals normally following cross-linking of BCR or CD40 or by stimulation with LPS (55).
B Cell Signal Transduction vs Antigen Trapping
The availability of embryonic stem (es) cells bearing a disrupted
Cr2 locus provided an opportunity to examine the mechanism of
complement enhancement of humoral immunity, i.e. B cell signal
transduction vs antigen trapping. Using the model of blastocyst
complementation in RAG-2-/- mice (71), Croix et al
constructed chimeric mice that expressed normal levels of
CD21/CD35 on their FDC, but their B cells (which were totally
derived from the Cr2-/- es cells) were negative (72).
Thus, the differential expression of CD21/CD35 on FDC and B cells
provided a model for testing the importance of the coreceptor
expression on B cells without apparent altering of
complement-mediated uptake of antigen by FDC. Despite expression
of normal levels of CD21/CD35 on their FDC, the Cr2-/-
chimeric mice had an impaired humoral response to the T-D antigen
NP-KLH. Therefore, it was concluded that CD21/CD35 expression by
B cells is essential in the formation of a normal secondary
response. It should be noted that splenic B cells in the RAG-2
mice reconstituted with Cr2-/- es cells expressed normal
levels of CD19 in the absence of CD21/CD35, thus confirming that
CD19 expression alone was not sufficient for the proposed
coreceptor enhancement. While these results suggested strongly
that complement-regulation of the B cell response was mediated by
the coreceptor, a combined requirement for trapping of antigen on
FDC and B cell response could not be ruled out. Alternatively,
Cr2+ B cells might be required for efficient transport of
antigen to the FDC.
These experiments also demonstrated that FDC in RAG2-/- mice are not inherently defective and appear to be normal following reconstitution with mature B and T cells. These results add to the growing evidence of the interdependency of B cells and FDC.
To examine further the importance of B cell coreceptor expression in humoral immunity, Fischer et al constructed BM chimeras between Cr2+ and Cr2- MHC-matched mice to obtain differential expression of CD21/CD35 on B cells and FDC (55). Reconstitution of lethally irradiated Cr2-/- mice with WT BM provided chimeric mice in which their B cells were derived from the donor (Cr2+), but the radio-resistant splenic FDC were of recipient origin (Cr2-/-). This phenotype, i.e. CD21/CD35+ B cells and CD21/CD35- FDC, was confirmed by immunohistochemical analysis and flow cytometry. Comparison of the immune response of chimeric mice (WT into Cr2-/-) with Cr2-/- and WT controls demonstrated that expression of CD21/CD35 on B cells was sufficient for an apparently normal secondary response to T-dependent antigens. However, it remains to be determined if the affinity of the antibody is the same as in WT mice. Despite an apparently normal GC reaction in immune chimeric mice, the level of antigen retained on CD21/CD35- FDC was dramatically reduced (MB Fischer, MC Carroll, unpublished results). Thus, it would not be unexpected to find that the long-term memory response of the chimeric mice is relatively weak.
Two Stages of B Cell Enhancement
The finding that coligation of the CD21/CD19 coreceptor complex
with the BCR enhanced B cell signal transduction led to the
"threshold hypothesis" (23, 24). According to this hypothesis,
the coreceptor is important for initial activation of naive or
virgin B cells bearing low-affinity surface IgM. Thus, the
coreceptor serves to lower the threshold such that antigens that
bind with low affinity to the BCR can induce sufficient signal to
activate the B cell. In the spleen, naive B cells encounter
T-dependent antigens within the white pulp and migrate into the
periarteriolar lymphoid sheath region (PALS) or T cell zone where
they interact with cognate helper T cells (73, 74). The outcome
of efficient activation of the B cell is development along two
pathways, i.e. antibody forming cells (AFC) within the PALS; and
entry into the follicles and initiation of a GC reaction. It is
not clear if progenitors of the same B cell clone can do both.
Formation of the AFC foci requires CD40L stimulation as treatment
with anti-CD40L at time of immunization blocks both AFC and GC
response (75). According to the threshold hypothesis, both
pathways of B cell development might be affected by absence of
coreceptor signaling. The primary antibody response to
bacteriophage X 174 is impaired in both guinea pigs (68) and
mice deficient in C3 or C4 (65). In addition, Cr2-/- mice
have an impaired primary response to bacteriophage (55). In
contrast, Molina et al. reported a normal primary response to
sheep red blood cell antigen (SRBC) in Cr2-/- mice (70).
It is probable that the structure of the antigen and the affinity
of preexisting B cells determine the necessity for complement in
the AFC foci response.
In an attempt to define at what stage/stages the coreceptor was involved in the early activation of B cells, the Cr2-/- and control mice were bred with mice (strain MD4) bearing the hen egg lysozyme (HEL)-specific immunoglobulin (Ig) heavy and light chain transgene (tg) (76). By varying the form of avian lysozyme used to activate the tg B cells in an adoptive transfer model, this system could be used to test the threshold hypothesis. Thus, this model could be used to determine directly if the affinity of the antigen affected the requirement for coreceptor expression. Two forms of lysozyme were selected that differ in relative binding affinity by at least 2000 fold, i.e. duck egg lysozyme (DEL) and turkey (TEL) that bind with relative affinities of 1x107 vs 2x1010 M-, respectively (77).
As expected, Cr2+ and Cr2- HEL-specific tg B cells respond similarly to DEL and TEL antigens in vitro; however, approximately 100-fold less TEL (high affinity antigen) than DEL (lower affinity antigen) is required to induce expression of activation markers CD86 (B7–2) and CD54 (ICAM-2) or to induce B cell proliferation in vitro (78). These in vitro experiments established that Cr2+ and Cr2- tg B cells respond similarly following antigen activation in the absence of complement ligand. However, a very different result is observed in vivo when complement is involved, as discussed below.
To examine the requirement of the coreceptor in vivo for antigens that bind with a relative lower affinity, Cr2+ and Cr2- tg B cells were mixed with DEL antigen and adoptively transferred into WT mice that had been immunized 7 days previously with the antigen. The advantage of this model is that the two groups of transgenic B cells can be compared in an ongoing humoral immune response in which the conditions of T help, antigen concentration, and FDC trapping are constant. In this model, WT mice were injected intravenously with 50 µg of soluble DEL without adjuvant 7 days prior to adoptive transfer and were not irradiated, so that the tg B cells were competing with endogenous B cells for antigen and T help. Although these conditions induce a less vigorous reaction than that found with adjuvants, this approach favors the requirement for natural immunity. Spleens were harvested 1–5 days following adoptive transfer and were analyzed by flow cytometry and immunohistochemistry. Day 1 following transfer, a similar number of Cr2+ and Cr2- tg B cells were identified within the splenic white pulp (both follicular and PALS regions). However, by day 5, very few, i.e. less than 2%, of the follicles included Cr2- tg B cells. In contrast, 65% of the follicles included Cr2+ tg B cells at the same time period. The level of surface expression of B cell activation markers CD54 and CD86 on day 1 after transfer was consistent with negligible activation of the Cr2- tg B cells. The impaired follicular retention of the Cr2- tg B cells and their low level of activation in vivo were consistent with the threshold hypothesis. Thus, in the absence of a functional coreceptor signaling, the Cr2- tg B cells were not sufficiently activated to compete with the endogenous B cells for limiting antigen and T help.
The relatively rapid loss of Cr2- HEL-specific tg B cells from the lymphoid compartment raises the question of whether they were eliminated or simply returned to circulation. If the Cr2- tg B cells simply failed to become activated on binding antigen, it might be reasoned they would remain within the follicles as they are when transferred into nonimmune recipients, i.e. approximately 50% of follicles include Cr2+ or Cr2- tg B cells 5 days after transfer (78). Thus, the lack of retention of Cr2- tg B cells within the follicles following transfer into DEL-immune recipients suggests that they are specifically eliminated from the lymphoid compartment. These results extend the threshold hypothesis and suggest that the coreceptor provides an essential survival signal following antigen binding that allows the antigen-specific B cell to continue along either the AFC pathway or entry into the follicular zone for initiation of the GC reaction (Figure 6a).
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Coreceptor-dependent retention within the follicular compartment can be overridden by very high affinity antigens. In contrast to that observed with DEL antigen, adoptive transfer of Cr2- HEL-specific tg B cells into TEL (high-affinity antigen) immune recipients leads to relatively high rates of occupancy of the follicles, i.e. approximately 65%. The increased survival of the Cr2- tg B cells within the follicles was not explained by a difference in the amount of T help or antigen trapping in the WT immune recipients because the immune response to DEL and TEL is similar in WT recipients. WT mice make a similar secondary response to the two forms of lysozyme as evidenced by secondary antibody titer, the number of splenic GC formed, and degree of T cell activation in an in vitro proliferation assay. The increased survival and follicular localization of the Cr2- tg B cells in the TEL vs the DEL immune recipients correlates with the findings in vitro that 100-fold less high-affinity than low-affinity antigen is required for B cell activation in vitro.
CD21 Ligand Promotes GC Survival
Examination of the survival of Cr2+ and Cr2-
HEL-specific tg B cells following transfer into WT mice immunized
with the high-affinity antigen TEL demonstrated a striking
difference in the number of tg B cells identified within the GC
regions, i.e. 6% vs 54% of GC occupied by Cr2- vs
Cr2+ tg B cells, respectively. Despite retention within
the follicles, Cr2- tg B cells failed to compete with
endogenous B cells for space within the GC. Two possible
explanations for this observation are that either the
Cr2- tg cells failed to enter the GC or they entered but
failed to survive. The former possibility seems unlikely since
Cr2- mice (as well as C3- and C4-deficient mice) can
initiate a GC, albeit at a reduced rate. Moreover, their size is
approximately 10-fold less than that of WT controls, and this
suggests limited survival of the GC B cell. Thus, irrespective of
the affinity of the antigen, B cell expression of CD21/CD35 is
required for survival within the GC. This finding was not
predicted by the threshold hypothesis and suggests that
coligation of CD21/CD19 coreceptor and BCR not only has an
enhancing effect but provides a qualitative signal.
The clonal selection hypothesis proposes that memory B cells are selected by antigen and only those that bind with the highest affinity survive (79, 80). Intense competition for antigen occurs within the specialized microenvironment of the GC. Here, B cells rapidly divide and acquire somatic mutations within their VH and VL region of rearranged Ig genes. Mutants that bind antigen with greater affinity are selected for entrance into the pool of memory cells. Although competition of GC B cells is antigen-dependent, additional signals are required for survival (81, 82). Soluble antigen alone is not sufficient to ensure survival and in fact can be deleterious. Flooding the GC with specific soluble antigen during the period of peak GC activity can result in a rapid elimination of the GC B cells some of which undergo apoptosis and others traffic to the PALS zone (83, 84, 85). Shokat & Goodnow proposed that B cell survival within the GC not only is antigen dependent but requires contact with the FDC surface, and this provides a survival signal (81, 84). Following FDC contact, GC B cells acquire antigen from the FDC and present it to the cognate T cell resulting in continued development into a memory cell. The requirement for T cell help was confirmed by the demonstration that blocking of CD40 ligand at the critical GC period has an effect similar to that of soluble antigen (75), i.e. elimination of GC. Thus, at least three signals are required for B cell survival within the GC: (a) antigen, (b) T help, and (c) FDC contact.
Antigen is retained on FDC via FcR and complement
receptors (5, 86). Immune complexes are putatively transported to
the FDC in a mechanism that is B cell dependent (81). Complement
is important in antigen deposition and possibly transport because
mice transiently depleted of C3 fail to efficiently retain
aggregated IgG on their FDC (4, 5). CD21 and CD35 receptors are
the major complement receptors on FDC, and deficiency in these
receptors results in a dramatic reduction in the retention of
antigen in immune animals (MB Fischer, S Goerg, M Ma, MC Carroll,
unpublished results). A hypothesis that would explain the role of
complement in the GC response is that the B cell coreceptor is
essential for FDC contact and delivery of a survival
signal (Figure 6b). Support for this hypothesis comes not only from the
adoptive transfer experiments discussed above but from the
finding that FDC coated with C3d and immune complexes support
long-term survival of B cells activated by either LPS or antigen
plus T help in vitro (87). Interestingly, in the in vitro assay,
the survival effect of FDC can be blocked by addition of sCR2 or
anti-CD21 antibody or when either the B cell or FDC are deficient
in CD21/CD35 (87). Thus, long-term survival of activated B cells
in this in vitro model depends on contact between the CD21 ligand
on the FDC and CD21 receptor on the B cell. A prediction of this
model is that injecting immune mice with sCR2 or C3d at the
optimal GC period would eliminate the GC in a manner similar to
that observed with soluble antigen (83, 84, 85) or anti-CD40
ligand treatment (75). Using human tonsiler B cells bearing a GC
phenotype (CD38hi, CD20hi, CD10+,
CD21+CD19+, CD23lo, sIgMlo,
IgD-, and CD14-), Bonnefoy et al find that a
subset of CD21-specific antibodies can rescue greater than 40% of
the cells from apoptosis in vitro (88). However, they argue that
the important CD21 ligand is CD23 because soluble CD23 can also
block apoptosis. The role of human CD23 appears to differ between
mice and humans as mice deficient in CD23 have normal GC
responses and mouse CD23 does not appear to bind to mouse CD21.
In summary, adoptive transfer experiments not only confirm the threshold hypothesis in vivo but extend it to demonstrate that the coreceptor provides a critical survival signal for naive B cells on encounter of low-affinity antigen. Further, they demonstrate that CD21-CD21 ligand interaction provides an essential survival signal for GC B cells in vivo irrespective of the affinity of the antigen.
Setting the Threshold for B Cell Tolerance
An important component of the clonal selection theory of Burnet
is that autoreactive clones of lymphocytes are eliminated on
encounter with self-antigens (79). Strength of BCR signal is a
major factor in induction of B cell tolerance in the peripheral
lymphoid compartment just as it is in B cell activation (reviewed
in 89). The study of the anti-HEL/soluble HEL double tg mice has
led to the hypothesis that strength of BCR signal determines
whether self-reactive B cells are deleted, anergized, or ignored.
Expression of a membrane form of HEL leads to clonal deletion of
HEL-specific B cells within BM, whereas expression of a soluble
form of HEL (sHEL) leads to clonal anergy in the periphery. Thus,
by varying the serum concentration of sHEL and the affinity of
the HEL-specific BCR, it was determined that induction of
peripheral tolerance was dependent on the overall strength of the
BCR signal.
A link between complement and autoimmunity is suggested by the observation that a major risk factor for systemic lupus erythematosus (SLE) in humans is genetic deficiency in the early components of the classical pathway of complement, i.e. Clq, Clr, Cls, C2, or C4A and C4B (90). In addition, partial deficiency in C4, i.e. null allele for C4A isotype, is an apparent risk factor for SLE (91). Given the role of complement in clearance of immune complexes, one explanation for the increased risk of SLE among complement-deficient individuals is increased accumulation of immune complexes that in turn enhance the pathology of autoimmune disease. However, this explanation does not account for the initiation of autoantibody formation. An alternative explanation is a corollary to the threshold hypothesis. Accordingly, signaling via the BCR is enhanced by the coreceptor irrespective of whether the antigen is foreign or self as long as the antigen is coated with C3d. Thus, it is speculated that a subset of self-antigens (as proposed for environmental antigens) are recognized by natural antibody resulting in attachment of C3d (as found in reperfusion injury). The encounter of naive self-reactive B cells with self-antigen coupled to C3d (or C4d) ligand cross-links the coreceptor and BCR delivering an enhanced tolerogenic signal (in the absence of T-help). Thus, in the case of complement-deficient individuals, self-reactive B cells encountering specific antigen in the peripheral lymphoid compartment receive a subthreshold signal in the absence of coreceptor cross-linking, and the clone remains in circulation. A secondary event resulting in T help and release of sufficient levels of self-antigen to overcome the increased threshold in activation would activate the self-reactive clones, resulting in release of autoantibody. Thus, it is proposed that deficiency in early complement components would result in both a reduction in the efficiency of targeting of self-antigens to the lymphoid compartment and an absence of CD21 ligand for coreceptor enhancement of signaling and therefore lead to an accumulation of self-reactive B cell clones.
To test this hypothesis, Goerg et al have bred Cr2-/- or C4-/- mice with the B6 lpr/lpr congenic strain (92). The latter develop a mild form of lupus-like disease characterized by late-onset of lymphadenopathy and autoantibodies against nuclear antigens (ANA), double-strand DNA (dsDNA), and IgG rheumatoid factor (Rh factor). It was predicted that deficiency in either the coreceptor (Cr2-/-/lpr/lpr) or its ligand (C4-/-/lpr/lpr) would exacerbate autoimmune disease as elimination of self-reactive B cells would be impaired to even a greater extent than in the fas-defective mice (B6 lpr/lpr). As predicted, deficiency in either CD21 or C4 in combination with lpr/lpr leads to more severe autoimmune disease. CD21-/-lpr/lpr mice have significantly higher autoantibody titers (ANA, anti-dsDNA and Rh factor) and autoimmunity occurs at an earlier age than in the WT/lpr/lpr controls (92). These results support the hypothesis that regulation of self-reactive B cells is altered by the absence of CD21/CD35 expression. Similarly, ANA autoantibody titers appear sooner (by 10 weeks of age) and are significantly higher in C4-lpr/lpr than in C4+lpr/lpr controls. Interestingly, both groups of deficient mice, i.e. CD21-/- and C4-/-, mice clear IgG immune complexes from the vasculature similar to that of normal mice. Thus, impairment of immune clearance of IgG containing complexes would not explain the increased susceptibility to lupus-like disease in complement-deficient mice. It will be important to examine the complement-deficient mice in more defined Ig transgenic models such as anti-HEL/sHEL (93) or anti-DNA tg mice (94).
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CONCLUDING REMARKS |
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While the focus of the review has been on the importance of CD21 as a coreceptor on B cells, its role in antigen retention on FDCs should not be overlooked. Studies in vitro of the interaction between B cells and FDC confirm the importance of C3d ligand for functional contact and delivery of a survival signal to the B cell (87). The model that is developing is that B cells like T cells recognize antigen that is bound to an accessory cell (FDC in case of B cell), and this contact provides a signal through the antigen receptor and the coreceptor (Figure 2).
Finally, new results suggesting a role for complement in regulation of self-reactive B cells and clonal selection of B-1 cells support a broader participation of complement in adaptive immunity than was previously held. It will be important to examine the repertoire of the complement-deficient strains to look for alterations in their preimmune B cell repertoire that would explain their predisposition to autoimmunity and absence of specific natural antibodies.
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ACKNOWLEDGMENTS |
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I would like to thank my collaborators Joe Ahearn, Stephen Galli, Chris Goodnow, Herb Hechtman, Garnett Kelsoe, Francis Moore, Jr., and John Tew who participated in many of the experiments discussed in the review. I would also like to thank the members of my laboratory who contributed to the experiments and in construction of the knockout mice: Michael B. Fischer, Siegfried Goerg, Minghe Ma, Andrey Prodeus, Russell Reid, Li-Ming Shen, Junrong Xia, and Xiaoning Zhou. Michael Carroll is supported by grants from the National Institutes of Health, the Lupus Foundation, and Cambridge Antibodies Technology.
Annu. Rev. Immunol. 1998. 16:545-568
Copyright © 1998 by Annual Reviews. All rights reserved
0732-0582/98/0410-0545
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