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The Journal of Immunology, 00, 164: 4522-4532.
Copyright © 00 by The American Association of Immunologists

Humoral Immune Responses in Cr2-/- Mice: Enhanced Affinity Maturation but Impaired Antibody Persistence1

Zhibin Chen*, Sergei B. Koralov*, Mariya Gendelman*, Michael C. Carroll{dagger} and Garnett Kelsoe2,*

* Department of Immunology, Duke University Medical Center, Durham, NC 27710; and {dagger} Center for Blood Research, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The discovery of complement receptors on B cells by Nussenzweig and colleagues (1, 2, 3) suggested that the complement system might affect acquired immunity. Pepys (4, 5) tested this possibility by transient depletion of C3 in mice with cobra venom factor and observed suppression of Ab responses to T-dependent Ags. Subsequent studies in guinea pigs (6, 7, 8, 9), dogs (10), humans (11), and mice (12) demonstrated that genetic deficiencies in C2, C3, or C4 resulted in similar immunological defects. Recently, Dempsey et al. (13) generated T-dependent Ags comprised of hen egg lysozyme and two or three tandemly aligned copies of C3d. These novel Ags were 1,000- to 10,000-fold more immunogenic in mice than the unmodified hen egg lysozyme, dramatically illustrating the importance of complement in enhancing Ab responses.

The effect of complement on B cell responses is thought to be mediated by two distinct complement receptors, CD21 (CR2) and CD35 (CR1), that are expressed on B cells and follicular dendritic cells (FDCs).3 Administration of a rat mAb specific for both CD21 and CD35 suppressed primary Ab responses of mice to T-dependent Ags, whereas an Ab specific for CD35 alone had a more modest immunosuppressive effect (14, 15, 16). This suggested that CD21 may play a more prominent role in mediating complement’s immune-enhancing effects than does CD35. Supporting this argument, a fusion protein containing the complement-binding domains of human CD21 suppressed primary Ab responses to T-dependent Ags in mice (17). Two mechanisms have been proposed to explain enhancement of humoral immunity by CD21 (18, 19): facilitation of Ag retention by FDC in germinal centers (GCs) (20, 21, 22, 23) and enhanced recruitment of the CD21/CD19/CD81 coreceptor into the B cell Ag receptor (BCR) complex (24, 25, 26, 27). Ag bound to FDCs drives the GC reaction that generates the B cell memory compartment, whereas the CD19/CD21 coreceptor lowers B cell activation thresholds.

Several groups have used gene disruption to clarify the role(s) of CD21/35 in immunity (28, 29, 30). In mice, CD21 and CD35 are alternatively spliced gene products encoded by the Cr2 locus (31, 32, 33). Disruption of the Cr2 loci abolishes expression of both CD21 and CD35 and results in impaired humoral immune responses (28, 29). However, the degree of impairment is controversial. One line of Cr2-/- mice failed to generate serum Ab responses to immunization with 3 x 107 PFU of bacteriophage {Phi}X174 in the absence of adjuvant. Nonetheless, a 10-fold increase in Ag dose elicited Ab production but only to levels significantly below that of wild-type controls (28). Reconstitution of lethally irradiated Cr2-/- mice with bone marrow from MHC-matched Cr2+/+ donors repaired this defect, indicating that impaired Ab production was a defect of B cells (28). Supporting this notion, mice lacking CD21/35 only on B cells failed to generate Ab responses to 10 µg (4-hydroxy-3-nitrophenyl)acetyl (NP)-keyhole limpet hemocyanin in alum adjuvant (30). An independently generated line of Cr2-/- mice also displayed markedly reduced primary humoral responses but responded to both high and low doses of SRBCs (29). In addition, defective Ab responses were observed even when this line of Cr2-/- mice was reconstituted with Cr2+/+ bone marrow, suggesting a significant role for CD21/CD35 on FDCs (34).

CD21 has also been shown to promote the survival of GC B cells. Stimulation of human tonsillar GC B cells with anti-CD21 mAbs in vitro induced the expression of Bcl-2 and reduced levels of apoptosis (35, 36). Adoptively transferred, Ag-specific Cr2-/- B cells did not persist in the GCs of wild-type mice, despite expression of BCRs with high affinity for Ag (37). Nonetheless, in the absence of competition from CD21/CD35+ cells, B lymphocytes in Cr2-/- mice can form GCs (28, 29, 34), although the magnitude of the reaction is significantly reduced (28). The quality of the GC reaction in the absence of CD21/CD35, e.g., hypermutation, clonal selection and affinity maturation, and maintenance of B cell memory compartments, remains unknown.

To investigate further the function of CD21/CD35 in B cell responses, we bred the Cr2-/- mutation onto the Ighb genetic background of C57BL/6 mice to study immune responses to NP. In all inbred mouse strains carrying Ighb and Ig{lambda}1, primary Ab responses to immunogenic conjugates of NP are clonally restricted (38). Virtually all NP-specific Abs in Ighb mice bear the {lambda}1 light chain (39, 40) and use VH186.2 and DFL16.1 gene segments to encode the Ig heavy chain (41, 42, 43). This response is compartmentalized into pauciclonal foci of Ab-forming cells (AFCs) along the periarteriolar lymphoid sheath (PALS) and follicular GCs (42, 43, 44, 45). A common precursor(s) that is activated in the outer PALS establishes both populations (42). NP initially activates B cells expressing {lambda}1 light chain and heavy chains encoded by VH gene segments in the V186.2 and V3 subfamilies of the J558 VH homology group (44); as the primary response progresses, GC B cells carrying V186.2-to-DFL16.1 VDJ rearrangements (so-called canonical rearrangements) achieve dominance. By day 8 of the primary response, >=80% of {lambda}1+ GC B cells express rearrangements of V186.2, whereas the VH gene segments that are common in the early primary response, V23, C1H4, CH10, and 24.8 (analogues), become rare. By day 7–8, primary GC B cells acquire point mutations and are subject to a affinity-driven selection. Crippling mutations in VDJ rearrangements are common in day 8 GCs but are rare at days 14 and 16 after immunization. On the other hand, mutations that are known to increase the affinity to NP are more frequently observed in late GCs (43).

The GC reaction is necessary for long-lasting primary serum Ab responses. A single exposure to Ag elicits persistent AFCs in bone marrow (46, 47, 48, 49) and significant levels of Ab for as long as 120 days (48). Even after the GC reaction wanes, bone marrow AFCs undergo affinity-driven clonal selection, and the affinity of the serum Ab increases (48). The forces that drive these processes are unknown.

In this study we have investigated the primary Ab response and GC reaction to NP in Cr2-/- mice, to characterize the effects of Ag dose, the persistence and affinity of serum Ab, and the somatic genetics of the GC reaction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Cr2-/- (28) mice were originally established on 129/Sv genetic background. These mice were crossed with C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) and F2 offspring were typed at Cr2 loci and for Igh haplotype. The status of the Cr2 loci was determined by Southern blot analysis of DNA from tail tissue (28) and was verified by flow cytometric analysis of CD21/CD35 expression on peripheral blood B cells (see below). The Igh haplotype was determined by flow cytometry of peripheral blood B cells (see below) and confirmed by ELISA of serum IgM. More than 10 male and female F2 animals carrying at least one disrupted Cr2 allele on an Ighb/b background were selected for breeding to generate the F3 homozygous and heterozygous knockout mice used in this study. Heterozygote F3 (Cr2+/-) animals served as normal phenotype controls to ensure that any background genetic effects were distributed without regard to the disrupted gene loci. C57BL/6 mice were also used as normal controls. All mice were maintained under identical specific-pathogen-free conditions at the Duke University Medical Center Vivarium (Durham, NC) and were used at the age of 2–3 mo.

Ags and immunization

The succinic anhydride esters of NP (Genosys, Woodland, TX) were reacted with chicken gamma-globulin (CG) (Pel-Freeze Biological, Rogers, AR) or BSA (United States Biochemical, Cleveland, OH) as described (45). Hapten substitution ratios were determined spectrophotometrically. Mice were immunized with a single i.p. injection of NP12-CG precipitated in alum.

Quantification of serum NP-specific Abs

Serum IgM, IgG1, or {lambda}1 Ab specific for NP was quantified by ELISA on NP6-BSA or NP20-BSA (48). In brief, 96-well plates (Falcon 3912; Becton Dickinson, Oxnard, CA) were coated with 20 µg/ml NP-BSA in 0.1 M carbonate buffer (pH 8.8) at 4°C overnight and then were blocked and washed with PBS (pH7.4) containing 0.1% Tween 20 (Sigma, St. Louis, MO) and 1% BSA. Serially diluted sera were added to duplicate wells and incubated for 1 h at room temperature. Each plate included standard controls of serially diluted monoclonal IgG1/{lambda}1 Abs, H33L{gamma}1/{lambda}1, and pEVHC{gamma}1 (50). These Abs bind NP with different affinities (Ka {cong} 2.0 x 107 M-1 and 1 x 106 M-1, respectively). The IgM/{lambda}1 anti-NP Ab, B1-8 (Ka {cong} 1.0 x 106 M-1) (50), was used as a control to quantititate NP-specific serum IgM Ab. After washing, bound serum Ab was revealed by HRP-conjugated goat anti-mouse IgM or IgG1 (Southern Biotechnology Associates, Birmingham, AL) or biotinylated anti-{lambda}1 (Ls136) and then by HRP-conjugated streptavidin (Southern Biotechnology Associates). HRP activity was visualized using TMB peroxidase substrate kit (Bio-Rad Laboratory, Hercules, CA). After the reaction was stopped with 1N sulfuric acid, ODs were read at 450 nm on an Emax ELISA reader (Molecular Devices, Sunnyvale, CA) and analyzed with SOFTmax PRO software (Molecular Devices). Concentrations of serum Abs were determined by the comparison of titrated samples to standard curves.

Enzyme-linked immunospot (ELISPOT)

NP-specific AFCs from bone marrow were estimated by ELISPOT on NP20-BSA and NP6-BSA substrates as described (48).

Affinity measurements of anti-NP serum Abs

The affinity of anti-NP serum Ab was estimated by calculating the ratios of NP6-binding Ab to NP20-binding Ab (51). Fluorescence quenching (52, 53) was also performed to measure the Ka of pooled samples of serum Ab. Briefly, serum IgG was purified from the sera of four to eight mice using a protein G-Sepharose column (Amersham Pharmacia Biotech, Picataway, NJ); recovered IgG was adjusted to a concentration of 50 µg/ml in PBS containing 0.02% Tween 20. Fluorescence quench was titrated over a three-log range (10-8–10-5 M) of monovalent hapten (NP-caproate; Genosys). Nonspecific quenching by an irrelevant, dextran-specific Ab (IgG1), MOPC21 (ICN Pharmaceuticals, Costa Mesa, CA), was determined in each assay for the calculation of NP-specific Ka.

Immunohistochemistry

Six-micrometer-thick sections of frozen spleen were prepared (45) and stored at -80°C until use. Before staining, sections were rehydrated in PBS and blocked with PBS containing 10% FCS and 0.1% Tween 20. Hydrated sections were then stained in tandem with HRP-conjugated peanut agglutinin (PNA) (EY Laboratories, San Mateo, CA) and biotinylated Ls136 and then with streptavidin-alkaline phosphatase (Southern Biotechnology Associates) as described (45). Bound HRP and alkaline phosphatase activities were revealed with 3-amino-ethyl-carbazole (Sigma) and naphthol AS-MX phosphate/fast blue BB base (Sigma), respectively.

Flow cytometric analyses

Expression of CD21/CD35, IgMa, and IgMb on peripheral blood B cells was determined by flow cytometry. In brief, {cong}100 µl of blood was taken from the tail vein of individual mice; PBMCs were isolated from these samples over Lympholyte-M (Accurate Chemical and Scientific, Westbury, NY) density gradients. After washes with PBS containing 2% FCS and 0.08% sodium azide, cells were incubated with anti-Fc{gamma}RI/RII Ab (PharMingen, San Diego, CA) to block FcR-mediated binding. PBMCs were then stained with FITC-labeled anti-CD21/35 (PharMingen), PE-conjugated anti-IgMa (PharMingen), and biotinylated anti-IgMb (clone AF6-78) plus Tri-color- or Red 613-labeled streptavidin (Caltag, Burlingame, CA, and Life Technologies, Gaithersburg, MD, respectively).

To quantify splenic GC reactions by flow cytometry (54), splenocyte suspensions were depleted of RBCs cells by incubation in 0.83% NH4Cl and washed as above. After blocking FcR-mediated binding, cells were stained with FITC-labeled GL-7 (PharMingen), PE-conjugated anti-B220 (PharMingen), and 7-aminoactinomycin D (7-AAD; Molecular Probes, Eugene, OR) for 30 min.

Amplification and sequencing of VDJ rearrangements from single GCs

Cellular material ({cong}20 cells) was microdissected from individual {lambda}1+ PNA+ GCs identified by immunohistochemistry (see above) and digested with proteinase K as described (42, 43, 44). After heat inactivation, the reaction mixture was subjected to two rounds of PCR amplification (42, 43, 44) with Pfu polymerase (Strategene, La Jolla, CA). Briefly, the first round of 40 amplification cycles used primers homologous to the genomic DNA 5' of the transcription start site of the V186.2 VH gene segment and to a region in the JH2–JH3 intron. Two-microliter aliquots of this reaction mixture were reamplified for another 40 cycles using a second set of nested primers complementary to the first 20 nucleotides of the V186.2 and to the terminal portion of JH2 segment. Both 5' primers are complementary to VH gene segments in the V186.2 and V3 subgroups of the J558 VH family. Amplified VDJ DNA was purified with the QIAquick PCR Purification kit (Qiagen, Valencia, CA), digested with the BamHI and PstI restriction enzymes (New England Biolabs, Beverly, MA), and cloned into pBluescript SK+ (Strategene). Plasmid DNA from randomly picked clones of bacterial transformants was sequenced using an ABI 377 PRISM DNA sequencer with the Perkin-Elmer Dye Terminator Sequencing system (PE Biosystems, Foster City, CA).

Estimating frequencies of VH186.2 rearrangements in naive B cells

Splenic cells from three C57BL/6, Cr2+/-, or Cr2-/- mice (8 wk old) were pooled and stained with monoclonal anti-IgD-FITC (PharMingen), anti-IgMb-PE (PharMingen), and Ls136-biotin plus Red 613-labeled streptavidin (Life Technologies). IgM+IgD+{lambda}1+ cells were isolated by FACS and subjected to proteinase K digestion, PCR amplification, and cloning, as was done for microdissected GC B cells. Transformed bacterial clones were subject to colony hybridization using a VH186.2-specific probe and a framework 1-binding probe that hybridizes to most members of the VH186.2 and V3 VH subgroups (45, 55). The hybridization was repeated to screen 170–255 bacterial clones from each group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose dependence of serum Ab responses in Cr2-/- mice

Cr2-/- and C57BL/6 mice were immunized i.p. with 5, 10, 20, or 50 µg of NP-CG in alum. NP-specific serum IgM and IgG1 responses were determined by ELISA 10 days later (Fig. 1Go, a and b). With alum adjuvant, Cr2-/- mice mounted significant IgM and IgG1 responses at Ag doses as low as 5 µg. However, the responses of Cr2-/- mice were substantially less than those of Cr2+/- and C57BL/6 controls; 5 µg NP-CG elicited 3- and 4-fold lower IgM and IgG1 responses, respectively, in Cr2-/- mice than in either control group (Fig. 1Go, a and b). Increasing Ag dose to 50 µg reduced the deficit in Cr2-/- mice so that IgM Ab levels became equivalent, and IgG1 rose in Cr2-/- to half of that observed in C57BL/6 and Cr2+/- mice. In Cr2-/- mice, a 10-fold increase in Ag (from 5 to 50 µg) resulted in a 3-fold increase in IgM responses and a 7-fold increase in IgG1 responses. In Cr2+/- and C57BL/6 controls, Ag-specific IgM titers did not increase and IgG1 levels increased about 4-fold over the same dose range (Fig. 1Go, a and b). Similar Ag-dose responses were observed for total {lambda}1+ anti-NP serum Ab in Cr2-/- and control mice (Fig. 1Goc).



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  		FIGURE 1. Serum Ab responses (a–c) and GC reactions (d) to different doses of Ag in Cr2-/- (filled bars), Cr2+/- (stippled bars), and C57BL/6 (open bars) mice. Mice were immunized with 5–50 µg of NP12-CG precipitated in alum and sacrificed at day 10 postimmunization. NP-specific serum IgM (a), IgG1 (b), and {lambda}1 (c) Ab responses were quantified by ELISA. The GC reaction was evaluated (d) in individual mice by immunohistochemical staining with PNA. GCs were enumerated by microscopy in two distant sections for each spleen. Each bar represents the mean (±SEM) numbers of GCs per section from three to nine mice.

 
Serum Ab levels fall more rapidly in Cr2-/- mice

Single immunizations with NP-CG elicit long-lasting serum Ab that is produced by AFCs in the bone marrow (48). To follow the kinetics of Ab responses in the absence of CD21/CD35, Cr2-/-, Cr2+/-, and C57BL/6 mice were immunized with 50 µg NP-CG in alum and bled at days 3, 6, 10, 16, 30, 50, 70, 90, and 112 (Cr2+/- at days 6, 10, 30, 70, and 112) after immunization. NP-specific serum IgM and IgG1 Ab was below 0.13 µg/ml at day 3 postimmunization in both Cr2-/- and C57BL/6 mice. In Cr2+/- and C57BL/6 controls, IgM responses were first observed at day 6 (5.5 ± 0.9 and 5.4 ± 0.7 µg/ml ( ± SEM), respectively), peaked at day 10 (14.6 ± 1.3 and 18 ± 1.4 µg/ml, respectively), and then decayed about 10-fold by day 30 (1.8 ± 0.1 and 1.4 ± 0.10 µg/ml, respectively). Cr2-/- mice had NP-specific IgM levels (8.3 ± 2.1 µg/ml) not less than those of Cr2+/- and C57BL/6 controls at day 6. After day 6 of the response, serum IgM responses in the majority of Cr2-/- mice decreased more rapidly than controls did (day 10, 9.9 ± 2.1 µg/ml; day 30, 0.3 ± 0.1 µg/ml) (Fig. 2Goa). In Cr2-/-, Cr2+/-, and C57BL/6 mice, serum anti-NP IgG1 increased about 15-fold from day 6 to a peak at day 10. By day 16, serum anti-NP IgG1 levels were about 60% of day 10 values in both knockout and control animals. At later times, the rate of loss of specific IgG1 from the serum diverged between Cr2-/- and C57BL/6 or Cr2+/- controls (Fig. 2Go, b and c). Linear regression analysis on the mean logarithmic values of NP-specific serum IgG1 concentration (days 16–112) indicated that Ab responses decayed twice as fast in Cr2-/- mice (y = -0.012x + 2.726, r = 0.993) as in C57BL/6 controls (y = -0.006x + 3.157, r = 0.964) and Cr2+/- mice (y = -0.006x + 3.089, r = 0.956). The difference of slopes between C57BL/6 and Cr2-/- mice is statistically significant (p < 0.001, Student’s t test for the homogeneity of regression; Fig. 2Goc). In addition, regression analysis for Ab decay in individual mice demonstrated that the decay rates in C57BL/6 and Cr2+/- (slopes 0.006 ± 0.001, both) differ significantly from those in Cr2-/- animals (0.012 ± 0.002; p < 0.05, Student’s t test) but not from each other. These decay rates are far in excess of the half-life of passively injected IgG1 (56). Thus, the more rapid loss of serum IgG1 in Cr2-/- animals in comparison to Cr2+/- and C57BL/6 mice likely reflects declining Ab production rather than altered Ig catabolism.



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  		FIGURE 2. Accelerated decay of Ab responses in Cr2-/- mice. Mice were immunized with 50 µg of NP12-CG precipitated in alum. The serum levels of NP-specific IgM (a; days 3–30 postimmunization) and IgG1 (b; days 3–112 postimmunization) in individual mice were followed. Each line (•, Cr2-/-; {circ}, C57BL/6) represents the kinetics of an individual mouse. Linear regression analysis of mean logarithmic values (±SEM) of IgG1 levels (c) in Cr2-/-({blacksquare}), Cr2+/- ({diamondsuit}), and C57BL/6({square}) mice (four to eight in each group) from day 16 to day 112 after immunization reveals distinct rates of Ab decay.

 
Fewer NP-specific Ab-forming cells in the bone marrow of Cr2-/- mice late after immunization

Persistent IgG1 serum Ab is maintained by long-lived AFCs that reside in the bone marrow (46, 47, 48, 49). These bone marrow AFCs are present in Cr2-/-, Cr2+/-, and C57BL/6 mice in comparable numbers early in the primary response (day 14), but 62 days after immunization, the bone marrow of Cr2-/- mice contains only about half as many NP-specific, IgG1 AFCs as do controls (Fig. 3Go). The difference late in the response is due less to declining numbers of AFCs in Cr2-/- mice than in the failure of this compartment to expand. In Cr2+/- and C57BL/6 mice, frequencies of total (Fig. 3Go) and high-affinity (hatched bars) NP-specific bone marrow AFCs increase ~2-fold to day 64. In contrast, the frequencies of all NP-specific AFCs in Cr2-/- mice (Fig. 3Goa) slightly decline over the same interval, falling from 57 ± 7 to 39 ± 14 AFCs per 106 bone marrow cells. Virtually all of this decline was confined to the lower-affinity compartment of AFCs; frequencies of high-affinity AFCs in the bone marrow of Cr2-/- mice (Fig. 3Goa) remained unchanged from day 14 to day 62 of the response. In contrast, bone marrow AFC numbers and affinities were virtually identical in C57BL/6 and Cr2+/- mice (Fig. 3Gob).



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  		FIGURE 3. Frequencies of NP-specific, IgG1 AFCs in the bone marrow of C57BL/6 vs Cr2-/- mice (a) and of C57BL/6 vs Cr2+/- mice (b) early and late after immunization. Complete bars represent the mean frequencies (±SEM) of AFCs detected by ELISPOT on NP20-BSA substrates (low- + high-affinity cells), and hatched regions represent AFCs detected on NP6-BSA (high-affinity cells); data are from groups of four mice. *, p < 0.05, Student’s t test.

 
Enhanced affinity maturation in serum Ab of Cr2-/- mice

The affinity of anti-NP serum Ab was estimated by calculating the ratios of Ab bound to NP6-BSA vs NP20-BSA (51). From day 6 to day 10 postimmunization, NP6/NP20 binding ratios for Cr2-/-, Cr2+/-, and C57BL/6 mice increased 4- to 5-fold to reach similar averages by day 16. However, from day 30 to day 112, average NP6/NP20 binding ratios for Cr2-/- mice were always greater than those of Cr2+/- and C57BL/6 mice, indicating increased affinity. Affinity maturation in Cr2+/- and C57BL/6 mice was virtually identical (Fig. 4Goa).



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  		FIGURE 4. Enhanced affinity maturation of serum Ab in the absence of CD21/CD35. Serum samples (as in Fig. 2Go) from Cr2-/- ({blacksquare}), Cr2+/- ({diamondsuit}), and C57BL/6 ({square}) mice were titrated in ELISA (a) for the amount of high-affinity (binds NP6-BSA) vs total (binds NP20-BSA) NP-reactive IgG1. NP6/NP20 ratios were calculated and averaged for each group at each time point. Data represent the mean ratios (±SEM) exhibited by four to eight mice. In an independent experiment (b), cohorts of four to eight mice were immunized with 50 µg of NP12-CG in alum and sacrificed at various times after immunization; sera from mice in each group were pooled and IgG was purified from each pool. Fluorescence quenching assays were performed to measure the equilibrium Ka of the purified IgG for NP-caproate (48 ).

 
In an independent experiment, groups of Cr2-/- and C57BL/6 mice (n = 4–8) were killed at different times after immunization, and IgG Ab was purified from the pooled sera. Fluorescence quenching assays were performed to measure the average Ka for NP-caproate (50, 52, 53). Consistent with the relative affinity estimates by ELISA (Fig. 4Goa), the Ka of serum IgG for NP at day 10 postimmunization were virtually identical in Cr2-/- and C57BL/6 mice. By 28 days after immunization, the Ka of Ab from Cr2-/- mice was slightly higher than that of controls, but by day 59, the Ka of Ab from Cr2-/- mice was >=4-fold greater than that of C57BL/6 mice (Fig. 4Gob). These measured Ka confirm the ELISA affinity estimates of individual sera and demonstrate enhanced affinity maturation in the absence of CD21/35.

Cr2-/- mice exhibit reduced GC reactions at low doses of Ag

CD21/CD35 affect B cell persistence in GCs (37). Thus, we evaluated the ability of Cr2-/- mice to form GCs after immunization with different doses of Ag. Cr2-/- mice injected with 5 µg of NP-CG have ~5-fold fewer GCs than do C57BL/6 or Cr2+/- controls (Fig. 1God); the size of GCs in Cr2-/- animals was also reduced in comparison to controls (data not shown). Larger doses of Ag increased both the size and number of GCs in Cr2-/- mice so that at 50 µg of NP-CG in alum, GC reactions were comparable in Cr2-/- and C57BL/6 mice (Fig. 1God). Surprisingly, and in contrast to the Ag-dose-dependent increases of serum Ab (Fig. 1Go, a–c), the GC reaction of C57BL/6 mice was reduced at a dose of 50 µg (Fig. 1God). This reduction was significant in the minimization of differences between the GC responses of Cr2-/- and control mice at high Ag doses.

We studied the kinetics of the GC reaction in Cr2-/- mice by flow cytometric quantification of the B220+GL7+ population of GC B cells (54). At an Ag dose of 50 µg, the kinetics of GC reactions in Cr2-/- mice and C57BL/6 controls were very similar, with peak responses at day 10 and then monotonic declines to frequencies slightly above naive levels by day 28 (Fig. 5Go). This pattern of response is typical of normal responses (48).



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  		FIGURE 5. Kinetics of GC reactions in Cr2-/- ({blacksquare}) and C57BL/6 ({square}) mice are similar. Mice were immunized with 50 µg of NP12-CG in alum and sacrificed at different time points after immunization. Spleen cells were analyzed with flow cytometry for the numbers of B220+GL-7+ cells. Data represent the mean frequencies (±SEM) of GC B cells present in three to eight mice.

 
VDJ hypermutation in the absence of CD21/CD35

Ig heavy chain gene rearrangements present in GC B cells from Cr2-/- and C57BL/6 mice were amplified by a specific PCR and sequenced (43); 79 and 61 VDJ sequences, respectively, were obtained from each group. Ratios of productive:nonproductive rearrangements were significantly lower in Cr2-/- mice (58:21) than in C57BL/6 controls (54:7). Consistent with previous reports (41, 42, 43, 48), in the 54 productive VDJ rearrangements sequenced from C57BL/6 controls, a large majority (46/54; 85%) used the V186.2 VH gene segment, and 70% (38/54) contained DFL16.1 (Table IGo). The YYGS or YYGN motifs optimal for NP-binding were present at the V-to-D junction in 31 (57%) of these VDJ fragments, and each V186.2 gene segment contained an average of 2.6 mutations (Fig. 5Go and Table IGo). To our surprise, fewer than half (26; 45%) of the 58 productive rearrangements sequenced from Cr2-/- mice contained V186.2, and exactly half (29/58) used DFL16.1. Nonetheless, YYG(S/N) junctional motifs were slightly more common than in controls (69% vs 57%; Table IGo), suggesting active clonal selection in the absence of CD21/CD35. Somatic mutation was not reduced in Cr2-/- mice; average mutation numbers in knockout mice were somewhat higher than in C57BL/6 mice (3.8 vs 2.6; p < 0.05, Student’s t test) (Fig. 5Go and Table IGo).


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  		Table I. Somatic genetics of {lambda}1+ GC B cells from Cr2-/- and C57BL/6 mice1

 
Lower numbers of VDJ rearrangements containing VH186.2 in GCs of Cr2 knockouts could reflect alterations of the GC reaction or the naive repertoire. Thus, we recovered VDJ rearrangements from naive (IgM+IgD+{lambda}1+) splenic B cells from Cr2-/-, Cr2+/-, and C57BL/6 mice by PCR amplification and cloning. Virtually all cloned rearrangements hybridized to a framework region probe that identifies the V3 and V186.2 VH subfamilies (98–100%, C57BL/6; 91–99%, Cr2+/-; 98–100%, Cr2-/-). However, within this hybridizing set, VH186.2 frequencies in C57BL/6 and Cr2+/- mice (29–32% and 27–31%, respectively) were 2- to 3-fold higher than that of Cr2-/- mice (11–14%). Thus, the Cr2-/- genotype modifies specific VDJ frequencies within the pool of naive, splenic B cells.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
B cell responses to T-dependent Ags are mediated by the Ag-specific BCR and a nonspecific coreceptor comprising CD21, CD19, and CD81 (18, 19, 57). The complement system, a major component of innate immunity, responds to pathogens by activation of the classic or alternative pathways; both activation pathways converge at C3 with the consequence of covalent deposition of C3b, which is rapidly converted to C3d on the pathogens’ surface (19, 58). C3d-decorated Ag is thought to facilitate binding/retention by FDCs and the recruitment of the CD21 and coreceptor complex into close association with Ag-bound BCRs (18, 19, 57).

We studied the primary B cell responses to the NP hapten to dissect the role(s) of CD21/CD35 on serum Ab responses and the GC reaction. As shown in Fig. 1Go, reduced but significant serum IgM and IgG1 responses were elicited in Cr2-/- mice even by low doses of NP-CG in alum. This impairment was dose-dependent; increasing amounts of Ag gradually improved serum IgG1 Ab titers in Cr2-/- from ~25% to 50% of that in Cr2+/- and C57BL/6 controls (Fig. 1Gob). In a previous study, Cr2-/- mice failed to generate Ab responses to low numbers (3 x 107) of bacteriophage {Phi}X174 in the absence of adjuvant but mounted modest responses to a 10-fold increase in phage numbers (28). Similarly, high doses of Ag mitigated the suppression of humoral immune responses by anti-CD21/CD35 Ab treatment (14, 15). These observations contrast with a report (29) in which a 200-fold increase of Ag (SRBC) did not improve deficient primary IgG Ab responses in Cr2-/- mice. Differences in responsiveness by Cr2-/- mice correlate with the use of inflammatory adjuvants, agents known to mitigate other humoral deficiencies. Generally, our findings indicate that complement and its receptors enhance humoral immune responses to physiological (low) amounts of noninflammatory Ags, which is consistent with the concept that the CD21/CD19/CD81 coreceptor lowers the threshold for B cell activation (18, 19, 57).

Ag dose also affects the GC reaction. Consistent with earlier findings (28), the GC reaction of Cr2-/- mice was only about 35% of that in Cr2+/- and C57BL/6 controls at lower Ag doses (Fig. 1God). However, after immunization with 50 µg NP-CG in alum, Cr2-/- and C57BL/6 mice generated comparable numbers of splenic GCs and GC B cells (Figs. 1God and 5). The GC reaction in Cr2-/- mice given this amount of Ag is kinetically similar to wild-type responses (Fig. 5Go), but equalization of responses depended as much on a diminished GC response in C57BL/6 mice as it did on increased GC reactions in Cr2-/- animals (Fig. 1God). Decreased GC but not Ab responses in control animals are unexpected outcomes to higher Ag doses that merit further investigation. Reduced GC responses to 50 µg NP-CG in alum were not observed in mice deficient for CD21/CD35. Perhaps large quantities of Ag on FDCs synergize with coreceptor signals to limit the GC reaction (59, 60, 61).

VDJ hypermutation is intact in Cr2-/- mice. By day 10 postimmunization, significantly more VDJ mutations were present in the GC B cells of Cr2-/- mice than were those from C57BL/6 mice (Fig. 6Go and Table IGo). This increase possibly reflects more stringent selection. Prior work has shown that B cells lacking CD21/CD35 do not persist in wild-type GCs, even if they bear high-affinity BCRs (37). However, in the absence of normal B cell competitors, Cr2-/- B cells respond to abundant Ag/adjuvant with GCs that are quantitatively and qualitatively comparable to those of control mice (Figs. 1God and 5, Table IGo, and Refs. 28, 29). Thus, the advantage that is provided by CD21/CD35 to GC B cells must be relative rather than absolute. Amelioration of the deficient GC reaction of Cr2-/-mice by adjuvant and abundant Ag supports the role of CD21/CD35 in Ag retention by FDC/stromal cells (34). One function of the alum adjuvant is to serve as an Ag depot (62).




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  		FIGURE 6. Representative heavy-chain VDJ DNA sequences recovered from day 10 {lambda}1+ GCs of Cr2-/- (upper group) and C57BL/6 (lower group) mice. A total of 79 and 61 VDJ sequences were recovered from three Cr2-/- and two C57BL/6 mice, respectively. All unique VDJ sequences from both groups are presented. Each sequence is identified by a number (01–10) and lowercase letter (a–d) that follow the capitals "AB." The number identifies the GC origin, and the letter indicates the specific VDJ sequence. Sequences that share VDJ junctional regions presumably share clonal origins and are presented to show shared and unique mutations. VDJ rearangements were compared with germline VH186.2 and JH2 sequences; dashes indicate identity with these reference sequences. Sequences that overlap the 5' and 3' PCR primers are not shown. Underlined nucleotides are not found in homologous germline gene sequences available from GenBank, and may represent somatic mutation. "I" in 61AB03c between codons 52A and 53 indicates an insertion of 27 nucleotides (CTT GAG TGG ATT GGA AAT ATT AAT CCT), which is a repeat of the sequence from codons 45–52A. It is unknown whether this insertion resulted from an in vivo or in vitro process. Sequence data are available from GenBank under accession nos. AF146302-17 (C57BL/6) and AF146318-38 (Cr2-/-).

 
Our observations raise the question of how Ag is presented in GCs without CD21/CD35. In GCs, Ags are trapped primarily on the FDC surface in the form of Ag-Ab-complement complexes (20, 21, 22, 23). This Ag resource initiates and sustains the GC reaction. As Ag levels fall in GCs, increasingly intense selection for higher-affinity clones is believed to occur (38, 63, 64). Ag in GCs was thought to be retained on FDCs by Fc{gamma}R and CD21/CD35 (20, 21, 22, 23). However, targeted disruption of the common FcR {gamma}-chain gene increased rather than reduced Ag levels on FDCs (65). Similarly, mice deficient in Fc{gamma}RIIb have elevated Ab responses (66). In contrast, disruption of the Cr2 loci dramatically decreased Ag retention by FDCs (19, 34), indicating that CD21/CD35 play the primary role in holding Ag in GCs. In the absence of CD21/CD35, FDCs likely use the Fc{gamma}RIIb molecule as an alternative receptor for Ag retention. Fc{gamma}RIIb is expressed at lower levels than CD21/CD35 on FDCs and is thought to not bind IgM (67); these limitations in potential efficiency may be responsible for the strong dose dependence of the GC reaction in Cr2-/- animals (Fig. 1God).

In a milieu of reduced Ag on FDCs/stromal cells and in the absence of complete coreceptors, affinity-driven clonal selection could be more stringent in CD21/CD35-deficient mice. In fact, after day 30 of the response, the average affinity of serum Ab in Cr2-/- mice was significantly higher than that present in Cr2+/- and C57BL/6 controls (Fig. 4Go). At what site(s) is affinity maturation augmented in CD21/CD35-deficient mice? The activation of extra-follicular B cells in the spleens of Cr2-/- mice appears normal; initial Ab affinities are identical in Cr2-/- and control mice (Fig. 4Go), and early GC B cell populations in knockout animals were even more diverse than in controls (Fig. 5Go and Table IGo). These findings suggest that responding B cells in Cr2-/- mice expressed a broad range of BCR affinities and do not support predictions that limiting activation thresholds in the absence of CD21 restrict clonal diversity in immune responses.

Unusually intense selection may have taken place in the GCs of Cr2-/- mice. The average number of unique CDR3 sequences recovered from individual GCs was {approx}1 for both knockout and BL/6 mice. Ratios of replacement to silent mutations in the CDR2 of VDJ rearrangements were higher in Cr2-/- mice than in those recovered from C57BL/6 controls (Table IGo). However, given the decreased representation of canonical VH186.2/JH2 rearrangements present in Cr2-/- GCs (Table IGo), it is difficult to gauge selection intensity. Reduction in VH186.2 use in the GC B cells of Cr2-/- mice may simply reflect its relative rarity in naive repertoire. VH186.2/JH2 B cells are about 2- to 3-fold less frequent in {lambda}1+, naive splenic B cells from Cr2-/- mice than those from C57BL/6 and Cr2+/- mice. How CD21/CD35 alters the naive repertoire is unknown, but changes could reflect disturbed selection during B cell maturation (68). On the other hand, the remarkably high frequency of nonproductive VDJ rearrangements present in GCs of CD21/35-deficient mice might result from receptor revision in Ag-reactive cells (69). Nonetheless, B-cell clones expressing VH186.2/JH2 rearrangements are enriched in GCs of Cr2-/- mice to the same extent as for C57BL/6 mice. We do not know the affinity of noncanonical BCRs in Cr2-/- GC B cells. Despite their presence, the affinity of early serum Ab (day 16) is comparable in both C57BL/6 and Cr2-/- mice (Fig. 4Go), suggesting that not all noncanonical clones in Cr2-/- mice have low affinities to NP, even though noncanonical VDJ rearrangements from C57BL/6 GCs showed generally lower affinities (50). However, even if the starting affinities of noncanonical clones in Cr2-/- mice are low, they could achieve higher affinities by somatic mutation and selection in GCs (50). Selection is evident in the early (day 10) GCs of Cr2-/- mice (Table IGo and Fig. 6Go), but the phenotype of higher Ab affinity is not present until 30 days after immunization (Fig. 4Go). At this late stage of the B cell response, the GC reaction is ending/ended, but affinity-driven selection still shapes the long-lived AFC population of the bone marrow (48).

The persistence of serum Ab is reduced in Cr2-/- mice (Fig. 2Go), even when elicited by a dose of Ag that produces GC responses similar to those in controls (Fig. 5Go). In comparison to Cr2+/- and C57BL/6 controls, Cr2-/- mice initially generate similar levels of NP-specific serum IgM and 2-fold less IgG1 in response to 50 µg of NP-CG (Fig. 1Go, a and b). However, Ab titers decay twice as rapidly in Cr2-/- mice (Fig. 2Go). Associated with the accelerated loss of IgG1 Ab was enhanced affinity maturation (Fig. 4Go). Increased rates of Ab decay and affinity maturation mirrored preferential loss of bone marrow AFCs secreting lower-affinity IgG1 Ab (Fig. 3Go).

If CD21/CD35 play important roles in the GC reaction, why does affinity maturation of serum Ab appear to increase in their absence? As the kinetics of the GC reaction in Cr2-/- and C57BL/6 mice are similar at high Ag doses (Fig. 5Go), we propose that enhanced affinity maturation in Cr2-/- mice reflects selection of the AFCs in bone marrow or of their precursors. Although adoptive transfer experiments indicate that bone marrow AFCs do not depend on Ag for survival (49), we previously demonstrated that this population undergoes affinity maturation (Fig. 3Go) long after the GC reaction wanes (48). Thus, Ag plays a critical role in the fate of bone marrow AFCs, if only in their selection and not in their survival. In the absence of CD21/CD35, the bone marrow AFC population not only fails to expand but contracts by the preferential loss of lower-affinity cells (Fig. 3Goa). This selective contraction coincides with the conundrum of accelerated loss of serum IgG1 Ab levels and increased Ab affinity (Fig. 4Go).

Where and how Ags act on the bone marrow AFCs are unknown. Nonetheless, the accelerated loss of circulating Ab in Cr2-/- mice (Fig. 2Goc) suggests that Ag depots influence bone marrow AFCs and implies that this Ag may be detected/retained through a mechanism(s) involving CD21/CD35. For example, in the absence of CD21/CD35, Ag deposition and coreceptor signaling could result in more stringent clonal selection for the bone marrow AFC compartment. This might lead to the selective loss of lower-affinity clones and result in the enhanced affinity maturation of late IgG1 Ab. Indeed, a role for Ag dose on the rate of affinity maturation has long been recognized (70). In the first two weeks of primary Ab responses, Ab affinity is affected little by Ag dose, but at later times, guinea pigs and rabbits given low doses of Ag produce serum Abs with affinities manyfold higher than those of animals given higher Ag doses (71, 72). Thus, the phenotype of Cr2-/- mice may reflect reductions in available Ag. However, accelerated loss of serum Ab was not noted in earlier studies on Ag dose and affinity maturation (70, 71, 72), and our analogy may well be incomplete. An alternative explanation is that survival of bone marrow AFCs generally declines in the absence of persistent Ag stores. Increased Ab affinity in Cr2-/- mice would then reflect the survival of higher-affinity clones generated by unusually intense GC selection.

The effect of CD21/CD35 on the long-term Ab responses presents an interesting dilemma for humoral immunity to microbial infection. Does the complement system recruit and maintain a broad spectrum of specific AFC and memory B cells at the expense of optimal affinity maturation? Does increased Ab concentration balance more modest affinity? In vitro, the affinity/avidity of an Ab correlates with its ability to fix complement (73) and neutralize virus (74). However, in vivo tests of humoral protection from acute viral infection suggest that immunity by serum Ab depend solely on Ab concentration, given a minimal avidity threshold (74). It would be interesting to test the effect of CD21/CD35 deficiency on the longevity of humoral protection from viral pathogens. Presumably, the minimum serum concentration required for immunity would, over time, be compromised in Cr2-/- mice, but the residual Ab would exhibit enhanced (and compensating) affinity.


   Acknowledgments
 
We thank Dr. T. F. Tedder for helpful discussions and review of the manuscript and Lindsay Grey Cowell (North Carolina State University) for help with statistical analysis.


   Footnotes
 
1 This work was supported in part by U.S. Public Health Service Grants AI-24335 and AG-13789 (to G.K.), AI-39246, AI-36389 (to M.C.C.), and AG-10207 (to G.K. and M.C.C.). Back

2 Address correspondence and reprint requests to Dr. Garnett Kelsoe, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address: Back

3 Abbreviations used in this paper: FDC, follicular dendritic cell; GC, germinal center; BCR, B cell Ag receptor; NP, (4-hydroxy-3-nitrophenyl)acetyl; AFC, Ab-forming cell; CG, chicken gamma-globulin; ELISPOT, enzyme-linked immunospot; PNA, peanut agglutinin. Back

Received for publication September 14, 1999. Accepted for publication February 18, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lay, W. H., V. Nussenzweig. 1968. Receptors for complement on leukocytes. J. Exp. Med. 128:991.[Abstract]
  2. Bianco, C., R. Patrick, V. Nussenzweig. 1970. A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. I. Separation and characterization. J. Exp. Med. 132:702.[Abstract]
  3. Nussenzweig, V.. 1974. Receptors for immune complexes on lymphocytes. Adv. Immunol. 19:217.[Medline]
  4. Pepys, M. B.. 1972. Role of complement in induction of the allergic response. Nature New Biol. 237:157.[Medline]
  5. Pepys, M. B.. 1974. Role of complement in induction of antibody production in vivo: effect of cobra factor and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J. Exp. Med. 140:126.[Abstract]
  6. Ellman, L., I. Green, F. Judge, M. M. Frank. 1971. In vivo studies in C4-deficient guinea pigs. J. Exp. Med. 134:162.[Abstract]
  7. Ochs, H. D., R. J. Wedgwood, M. M. Frank, S. R. Heller, S. W. Hosea. 1983. The role of complement in the induction of antibody responses. Clin. Exp. Immunol. 53:208.[Medline]
  8. Bottger, E. C., S. Metzger, D. Bitter-Suermann, G. Stevenson, S. Kleindienst, R. Burger. 1986. Impaired humoral immune response in complement C3-deficient guinea pigs: absence of secondary antibody response. Eur. J. Immunol. 16:1231.[Medline]
  9. Bitter-Suermann, D., R. Burger. 1986. Guinea pigs deficient in C2, C4, C3 or the C3a receptor. Prog. Allergy. 39:134.[Medline]
  10. O’Neil, K. M., H. D. Ochs, S. R. Heller, L. C. Cork, J. M. Morris, J. A. Winkelstein. 1988. Role of C3 in humoral immunity. Defective antibody production in C3-deficient dogs. J. Immunol. 140:1939.[Abstract/Free Full Text]
  11. Jackson, C. G., H. D. Ochs, R. J. Wedgwood. 1979. Immune response of a patient with deficiency of the fourth component of complement and systemic lupus erythematosus. N. Engl. J. Med. 300:1124.[Abstract]
  12. Fischer, M. B., M. Ma, S. Goerg, X. Zhou, J. Xia, O. Finco, S. Han, G. Kelsoe, R. G. Howard, T. L. Rothstein, et al 1996. Regulation of the B cell response to T-dependent antigens by classical pathway complement. J. Immunol. 157:549.[Abstract]
  13. Dempsey, P. W., M. E. Allison, S. Akkaraju, C. C. Goodnow, D. T. Fearon. 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271:348.[Abstract]
  14. Heyman, B., E. J. Wiersma, T. Kinoshita. 1990. In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody. J. Exp. Med. 172:665.[Abstract/Free Full Text]
  15. Thyphronitis, G., T. Kinoshita, K. Inoue, J. E. Schweinle, G. C. Tsokos, E. S. Metcalf, F. D. Finkelman, J. E. Balow. 1991. Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo. J. Immunol. 147:224.[Abstract]
  16. Gustavsson, S., T. Kinoshita, B. Heyman. 1995. Antibodies to murine complement receptor 1 and 2 can inhibit the antibody response in vivo without inhibiting T helper cell induction. J. Immunol. 154:6524.[Abstract]
  17. Hebell, T., J. M. Ahearn, D. T. Fearon. 1991. Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254:102.[Abstract/Free Full Text]
  18. Fearon, D. T., R. H. Carter. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13:127.[Medline]
  19. Carroll, M. C.. 1998. The role of complement and complement receptors in induction and regulation of immunity. Annu. Rev. Immunol. 16:545.[Medline]
  20. Klaus, G. G., J. H. Humphrey, A. Kunkl, D. W. Dongworth. 1980. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53:3.[Medline]
  21. Mandel, T. E., R. P. Phipps, A. Abbot, J. G. Tew. 1980. The follicular dendritic cell: long term antigen retention during immunity. Immunol. Rev. 53:29.[Medline]
  22. Schriever, F., L. M. Nadler. 1992. The central role of follicular dendritic cells in lymphoid tissues. Adv. Immunol. 51:243.[Medline]
  23. Tew, J. G., J. Wu, D. Qin, S. Helm, G. F. Burton, A. K. Szakal. 1997. Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells. Immunol. Rev. 156:39.[Medline]
  24. Tuveson, D. A., J. M. Ahearn, A. K. Matsumoto, D. T. Fearon. 1991. Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex. J. Exp. Med. 173:1083.[Abstract/Free Full Text]
  25. Matsumoto, A. K., J. Kopicky-Burd, R. H. Carter, D. A. Tuveson, T. F. Tedder, D. T. Fearon. 1991. Intersection of the complement and immune systems: a signal transduction complex of the B lymphocyte-containing complement receptor type 2 and CD19. J. Exp. Med. 173:55.[Abstract/Free Full Text]
  26. Bradbury, L. E., G. S. Kansas, S. Levy, R. L. Evans, T. F. Tedder. 1992. The CD19/CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules. J. Immunol. 149:2841.[Abstract]
  27. Krop, I., A. L. Shaffer, D. T. Fearon, M. S. Schlissel. 1996. The signaling activity of murine CD19 is regulated during cell development. J. Immunol. 157:48.[Abstract]
  28. Ahearn, J. M., M. B. Fischer, D. Croix, S. Goerg, M. Ma, J. Xia, X. Zhou, R. G. Howard, T. L. Rothstein, M. C. Carroll. 1996. Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen. Immunity 4:251.[Medline]
  29. Molina, H., V. M. Holers, B. Li, Y. Fung, S. Mariathasan, J. Goellner, J. Strauss-Schoenberger, R. W. Karr, D. D. Chaplin. 1996. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proc. Natl. Acad. Sci. USA 93:3357.[Abstract/Free Full Text]
  30. Croix, D. A., J. M. Ahearn, A. M. Rosengard, S. Han, G. Kelsoe, M. Ma, M. C. Carroll. 1996. Antibody response to a T-dependent antigen requires B cell expression of complement receptors. J. Exp. Med. 183:1857.[Abstract/Free Full Text]
  31. Fingeroth, J. D., M. A. Benedict, D. N. Levy, J. L. Strominger. 1989. Identification of murine complement receptor type 2. Proc. Natl. Acad. Sci. USA 86:242.[Abstract/Free Full Text]
  32. Kurtz, C. B., E. O’Toole, S. M. Christensen, J. H. Weis. 1990. The murine complement receptor gene family. IV. Alternative splicing of Cr2 gene transcripts predicts two distinct gene products that share homologous domains with both human CR2 and CR1. J. Immunol. 144:3581.[Abstract]
  33. Molina, H., T. Kinoshita, K. Inoue, J. C. Carel, V. M. Holers. 1990. A molecular and immunochemical characterization of mouse CR2: evidence for a single gene model of mouse complement receptors 1 and 2. J. Immunol. 145:2974.[Abstract]
  34. Fang, Y., C. Xu, Y. X. Fu, V. M. Holers, H. Molina. 1998. Expression of complement receptors 1 and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific IgG response. J. Immunol. 160:5273.[Abstract/Free Full Text]
  35. Bonnefoy, J. Y., S. Henchoz, D. Hardie, M. J. Holder, J. Gordon. 1993. A subset of anti-CD21 antibodies promote the rescue of germinal center B cells from apoptosis. Eur. J. Immunol. 23:969.[Medline]
  36. Roberts, T., E. C. Snow. 1999. Cutting edge: recruitment of the CD19/CD21 coreceptor to B cell antigen receptor is required for antigen-mediated expression of Bcl-2 by resting and cycling hen egg lysozyme transgenic B cells. J. Immunol. 162:4377.[Abstract/Free Full Text]
  37. Fischer, M. B., S. Goerg, L. Shen, A. P. Prodeus, C. C. Goodnow, G. Kelsoe, M. C. Carroll. 1998. Dependence of germinal center B cells on expression of CD21/CD35 for survival. Science 280:582.[Abstract/Free Full Text]
  38. Kelsoe, G.. 1995. In situ studies of the germinal center reaction. Adv. Immunol. 60:267.[Medline]
  39. Makela, O., K. Karjalainen. 1977. Inherited immunoglobulin idiotypes of the mouse. Immunol. Rev. 34:119.[Medline]
  40. Reth, M., G. J. Hammerling, K. Rajewsky. 1978. Analysis of the repertoire of anti-NP antibodies in C57BL/6 mice by cell fusion. I. Characterization of antibody families in the primary and hyperimmune response. Eur. J. Immunol. 8:393.[Medline]
  41. Bothwell, A. L., M. Paskind, M. Reth, T. Imanishi-Kari, K. Rajewsky, D. Baltimore. 1981. Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a {gamma}2a variable region. Cell 24:625.[Medline]
  42. Jacob, J., G. Kelsoe. 1992. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. J. Exp. Med. 176:679.[Abstract/Free Full Text]
  43. Jacob, J., J. Przylepa, C. Miller, G. Kelsoe. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J. Exp. Med. 178:1293.[Abstract/Free Full Text]
  44. Jacob, J., G. Kelsoe, K. Rajewsky, U. Weiss. 1991. Intraclonal generation of antibody mutants in germinal centres. Nature 354:389.[Medline]
  45. Jacob, J., R. Kassir, G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173:1165.[Abstract/Free Full Text]
  46. Slifka, M. K., M. Matloubian, R. Ahmed. 1995. Bone marrow is a major site of long-term antibody production after acute viral infection. J. Virol. 69:1895.[Abstract]
  47. Manz, R. A., A. Thiel, A. Radbruch. 1997. Lifetime of plasma cells in the bone marrow. Nature 388:133.[Medline]
  48. Takahashi, Y., P. R. Dutta, D. M. Cerasoli, G. Kelsoe. 1998. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection. J. Exp. Med. 187:885.[Abstract/Free Full Text]
  49. Slifka, M. K., R. Antia, J. K. Whitmire, R. Ahmed. 1998. Humoral immunity due to long-lived plasma cells. Immunity 8:363.[Medline]
  50. Dal Porto, J. M., A. M. Haberman, M. J. Shlomchik, G. Kelsoe. 1998. Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers. J. Immunol. 161:5373.[Abstract/Free Full Text]
  51. Herzenberg, L. A., S. J. Black, T. Tokuhisa. 1980. Memory B cells at successive stages of differentiation: affinity maturation and the role of IgD receptors. J. Exp. Med. 151:1071.[Abstract/Free Full Text]
  52. Eisen, H. N., G. W. Siskind. 1964. Variations in affinities of antibodies during the immune response. Biochemistry 3:996.
  53. Jones, P. T., P. H. Dear, J. Foote, M. S. Neuberger, G. Winter. 1986. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321:522.[Medline]
  54. Han, S., B. Zheng, D. G. Schatz, E. Spanopoulou, G. Kelsoe. 1996. Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells. Science 274:2094.[Abstract/Free Full Text]
  55. Gu, H., D. Tarlinton, W. Muller, K. Rajewsky, I. Forster. 1991. Most peripheral B cells in mice are ligand selected. J. Exp. Med. 173:1357.[Abstract/Free Full Text]
  56. Vieira, P., K. Rajewsky. 1988. The half-lives of serum immunoglobulins in adult mice. Eur. J. Immunol. 18:313.[Medline]
  57. Tedder, T. F., M. Inaoki, S. Sato. 1997. The CD19-CD21 complex regulates signal transduction thresholds governing humoral immunity and autoimmunity. Immunity 6:107.[Medline]
  58. Fearon, D. T.. 1998. The complement system and adaptive immunity. Semin. Immunol. 10:355.[Medline]
  59. Pulendran, B., G. Kannourakis, S. Nouri, K. G. Smith, G. J. Nossal. 1995. Soluble antigen can cause enhanced apoptosis of germinal-centre B cells. Nature 375:331.[Medline]
  60. Shokat, K. M., C. C. Goodnow. 1995. Antigen-induced B-cell death and elimination during germinal-centre immune responses. Nature 375:334.[Medline]
  61. Han, S., B. Zheng, J. Dal Porto, G. Kelsoe. 1995. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. IV. Affinity-dependent, antigen-driven B cell apoptosis in germinal centers as a mechanism for maintaining self-tolerance. J. Exp. Med. 182:1635.[Abstract/Free Full Text]
  62. Gupta, R. K., B. E. Rost, E. Relyveld, G. R. Siber. 1995. Adjuvant properties of aluminum and calcium compounds. Pharm. Biotechnol. 6:229.[Medline]
  63. Kelsoe, G.. 1996. Life and death in germinal centers (redux). Immunity 4:107.[Medline]
  64. MacLennan, I. C.. 1994. Germinal centers. Annu. Rev. Immunol. 12:117.[Medline]
  65. Vora, K. A., J. V. Ravetch, T. Manser. 1997. Amplified follicular immune complex deposition in mice lacking the Fc receptor {gamma}-chain does not alter maturation of the B cell response. J. Immunol. 159:2116.[Abstract/Free Full Text]
  66. Takai, T., M. Ono, M. Hikida, H. Ohmori, J. V. Ravetch. 1996. Augmented humoral and anaphylactic responses in Fc{gamma}RII-deficient mice. Nature 379:346.[Medline]
  67. Unkeless, J. C., E. Scigliano, V. H. Freedman. 1988. Structure and function of human and murine receptors for IgG. Annu. Rev. Immunol. 6:251.[Medline]
  68. Prodeus, A. P., S. Goerg, L. M. Shen, O. O. Pozdnyakova, L. Chu, E. M. Alicot, C. C. Goodnow, M. C. Carroll. 1998. A critical role for complement in maintenance of self-tolerance. Immunity 9:721.[Medline]
  69. Hertz, M., V. Kouskoff, T. Nakamura, D. Nemazee. 1998. V(D)J recombinase induction in splenic B lymphocytes is inhibited by antigen-receptor signaling. Nature 394:292.[Medline]
  70. Goidl, E. A., W. E. Paul, G. W. Siskind, B. Benacerraf. 1968. The effect of antigen dose and time after immunization on the amount and affinity of anti-hapten antibody. J. Immunol. 100:371.[Abstract/Free Full Text]
  71. Siskind, G. W., P. Dunn, J. G. Walker. 1968. Studies on the control of antibody synthesis. II. Effect of antigen dose and of suppression by passive antibody on the affinity of antibody synthesized. J. Exp. Med. 127:55.[Abstract]
  72. Steiner, L. A., H. N. Eisen. 1967. Sequential changes in the relative affinity of antibodies synthesized during the immune response. J. Exp. Med. 126:1161.[Abstract]</