Deficiencies in C factors C2, C3, or C4 as well as lack of C receptors 1 and 2 (CR1/2) lead to impaired Ab production. Classical pathway activation plays a major role, as mice deficient in factor B, a key factor in the alternative pathway, have normal Ab production. Abs in complex with their specific Ag are known to feedback regulate the Ab response, and enhanced responses are initiated by IgM, IgE, and IgG. IgM acts via the C system, whereas IgE and IgG can operate independently of C via Fc receptors. Here we have investigated whether these isotypes are able to enhance Ab responses in mice lacking CR1/2. SRBC-specific IgM, administered with SRBC, does not enhance Ab responses in these animals. In contrast, 2,4,6-trinitrophenyl-specific IgE and IgG2a, administered with BSA-2,4,6-trinitrophenyl, induce potent Ab responses in CR1/2-deficient mice. Additionally, BSA administered with CFA or alum induced strong Ab responses in the absence of CR1/2. These results indicate that CR1/2 is needed to promote IgM-mediated induction of primary Ab responses. The data also show that the need for CR1/2 can be circumvented by Abs typical of a secondary immune response forming complexes with Ag or by conventional adjuvants, presumably mimicking physiological inflammatory reactions.

The C system plays a role in adaptive immunity in addition to its well-documented role in natural immunity. Animals genetically deficient in the early C components C2, C3, or C4 and animals treated with cobra venom factor (CVF)5 to deplete C activity exhibit impaired humoral immune responses to T-dependent and T-independent Ags (reviewed in Refs. 1 and 2). The receptor mediating the effects of C on Ab responses in vivo is most likely C receptor 2 (CR2 or CD21), which is predominantly expressed on B cells and can be found in association with CD19, an important coreceptor for B cell receptor (BCR)-mediated signaling (3). In mice, CR2 is derived from the same gene as complement receptor 1 (CR1 or CD35) via alternative splicing (4), with CR2 being the shorter gene product. For these reasons, no CR2-specific mAb or knockout mice exist, and distinction between the effects mediated by the two receptors is difficult. The treatment of mice with mAbs binding to CR1/2, but not with mAbs binding to CR1 alone (5) or treatment with a soluble CR2 molecule, competing with surface CR2 for ligand (6), led to severely impaired Ab responses to subsequent immunization. These findings imply that CR2 rather than CR1 is required for normal Ab production. Recently, mice deficient in CR1/2 (CR1/2−/−) due to gene targeting were shown to have poor humoral responses to T-dependent Ags (7, 8, 9, 10) as well as fewer and smaller germinal centers (GCs) (8, 9).

The need for C in initiation of Ab production is usually most pronounced when suboptimal Ag concentrations are used and can be partially overcome with optimal immunizations. For example, mice depleted of C by treatment with CVF produced near normal responses to Ag administered in CFA (11). Guinea pigs genetically deficient in C2 or C4 (12) as well as gene-targeted mice lacking C3 or C4 (13) produced significant Ab responses to high, but not to low, doses of bacteriophage ΦX174. It is not clear whether high doses of Ag can also overcome the need for CR1/2. In initial studies in which CR1/2 was blocked by mAbs, a strong Ab response was seen with optimal doses of erythrocytes, whereas suboptimal doses were unable to induce responses (5). However, using CR1/2−/− mice only marginal responses were detected even after repeated immunizations with high doses of Ag (7, 8, 9).

Whereas deficiencies in the classical pathway components C2 and C4 lead to impaired humoral responses, mice lacking factor B of the alternative pathway appear to have normal Ab responses (14). This implies that classical pathway C activation, known to be initiated by Ab/Ag complexes, is of major importance for the induction of a normal Ab response. Abs in complex with their specific Ag are known to have strong negative or positive feedback regulatory effects on humoral responses (15). Three isotypes can enhance Ab responses: IgG (16, 17, 18, 19), IgE (20, 21), and IgM (22, 23, 24). Enhancement by IgG can take place in the absence of C (18). IgE does not activate C, and IgE-mediated enhancement is known to be completely dependent on the low affinity receptor for IgE, CD23 (20, 21). However, a link between the C system and feedback regulation by Abs was seen in an experimental system in which IgM anti-SRBC administered together with suboptimal doses of SRBC enhanced the Ab response; IgM unable to activate C does not enhance, and normal IgM cannot enhance in C-depleted animals (24). In the present report we have investigated whether Ags in complex with IgM, IgG2a, or IgE as well as Ags administered with adjuvants are able to induce Ab responses in animals lacking CR1/2.

Male CR1/2−/− mice (H-2Ab) (7) were mated with female CBA/J mice (H-2Ak; Bommice, Bomholtgaard, Ry, Denmark). This was done to bring the CR1/2−/− mice to the H-2Ak haplotype, as H-2Ab mice are low responders to IgG and IgE immune complexes (25, 26). The F1 generation was intercrossed, and mice homozygous for the H-2Ak allele and mutant CR1/2 (CR1/2−/−) or wild-type CR1/2 (CR1/2+/+) were selected and used for breeding of CR1/2−/− and CR1/2+/+ mice. The offspring from these mice, 2–5 mo of age, matched for age and sex within each experiment, were used. Although the optimal strains would be fully congenic mice, the most important gene locus (I-A) for the studied Ab responses was similar in CR1/2−/− and CR1/2+/+ animals. Animals were bred in the animal facilities at the Department of Animal Development and Genetics, Uppsala University (Uppsala, Sweden). Female BALB/c mice (Bommice) were used in the experiments in which CR1/2 was blocked with anti-CR1/2 mAbs. These mice were kept at the animal facilities at the Biomedical Center, Uppsala University.

The H-2A haplotype (H-2Ak or H-2Ab) was analyzed by two independent PCR reactions. The Ak allele was detected using primers αK2 (5′-TTC CAA GTT GTG TTT TCC TG-3′) and αK1:2 (5′-TAT CAG TCT CCT GGA GAG ATT G-3′). The Ab allele was detected using primers αK1:2 (described above) and αB2 (5′-ACT CCC AAG TTG TGT TTT ACT A-3′). Gene amplification was performed in a 50-μl volume of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 0.2 mM dNTPs, 0.22 μM of primers αK2 and αB2, 0.28 μM of primer αK1, and 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT) for 25 cycles (95°C for 30 s; 55°C for 40 s; 72°C for 30 s). Both αK1:2/αK2 and αK1:2/αB2 give fragments 180 bp in size.

The CR1/2 genotype was analyzed by two independent PCR. The CR1/2−/− genotype was detected using the primer pair P3 (5′-CGC TGT TCT CCT CTT CCT CAT C-3′) and P4 (5′-GAT GGA TAC TTT CTC GGC AGG AGC-3′). The CR1/2+/+ genotype was detected using the primer pair P5 (5′-TGT CAG GCT CCT CCT AAA ATT ATC-3′) and P6 (5′-CTT TAC AAA GAC GGA TTT CTA TA-3′). Gene amplification was performed in a 50-μl volume of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 0.2 mM dNTPs, 0.22 μM each of primers (P3/P4 or P5/P6), and 0.5 U of AmpliTaq DNA polymerase (Perkin-Elmer/Cetus) for 35 cycles (94°C for 30 s; 65°C for 30 s; 72°C for 2 min (CR1/2−/− allele primer pair) or 94°C for 30 s; 55°C for 30 s; 72°C for 2 min (CR2+/+ allele primer pair)). The P3/P4 PCR gives a 400-bp DNA fragment, while the P5/P6 PCR gives a DNA fragment of 690 bp.

Rat IgG2b anti-mouse CR1/2 mAbs were derived from the hybridoma cell line 7G6 (27) and purified as previously described (5). The hybridoma cell lines IGELb4 (mouse IgE anti-2,4,6-trinitrophenyl (anti-TNP)) (28) and C4007B4 (7B4, mouse IgG2a anti-TNP) (17) were cultured in DMEM with 5% FCS. IgG2a was purified on a protein A-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s recommendations. IgE was purified by affinity chromatography on a Sepharose column conjugated with a rat anti-mouse κ mAb, 187.1.10 (29). Bound IgE was eluted with 0.1 M glycine-HCl, pH 2.8. Abs were dialyzed against PBS, sterile-filtered, and stored at −20°C. Protein concentrations were determined by absorbance at 280 nm, assuming that an absorbance of 1.5 equals 1 mg/ml of Ab. Polyclonal IgM anti-SRBC was prepared from sera obtained from mice 5 days after i.p. immunization with 10% SRBC in PBS. Sera were diluted 1:2 in 0.05 M sodium phosphate/0.15 M NaCl, pH 7.4, and isolated by fluid phase liquid chromatography gel filtration using Superdex 200 prep grade, High Load 26/60 (Amersham Pharmacia Biotech). The IgM-containing column fractions with reactivity to SRBC, as determined by ELISA (30), were pooled and concentrated using a P10 Amicon concentration filter (Amicon, Beverly, MA) according to the manufacturer’s instructions. The direct hemagglutination titer was determined as previously described (23).

OVA (grade V, A-5503), BSA (fraction V, A-3059), and TNP (picrylsulfonic acid/hydrate) were obtained from Sigma (St. Louis, MO). TNP was conjugated to BSA in 0.28 M cacodylate buffer, pH 6.9. After 70 min of incubation at room temperature the reaction was stopped by an excess of glycyl-glycin (1 mg/ml; Merck, Darmstadt, Germany). Proteins were dialyzed against PBS, sterile-filtered, and stored at 4°C. The number of TNP residues/BSA was determined as described previously (31), and a conjugate with 12 TNP/BSA was used. SRBC and horse erythrocytes (HRBC) were obtained from the National Veterinary Institute (Uppsala, Sweden) and stored in sterile Alsever’s solution at 4°C. Erythrocytes were prepared by washing three times in sterile PBS for use as Ags or in HBSS for use in plaque-forming cell (PFC) assays.

Mice were immunized in their tail veins with 0.1 ml of IgG2a anti-TNP/BSA-TNP or IgE anti-TNP/BSA-TNP complexes formed by incubating Ag and Abs together at 37°C for 1 h in PBS along with 20 μg of OVA (as a specificity control). In experiments involving SRBC and IgM, Ab was administered i.v. in 0.1 ml of PBS, followed after 1 h by SRBC and HRBC (as a specificity control) in 0.1 ml of PBS i.v. Equal volumes of CFA (Difco, Detroit, MI) and BSA were emulsified, and two 50-μl emulsions (20 μg of BSA/mouse) were given s.c. in each flank. Alum (Pierce, Rockford, IL) was emulsified with BSA as described by the manufacturer, and two 50-μl emulsions (20 μg of BSA/mouse) were given s.c. in each flank.

Mice were bled from the tail veins, and individual sera were analyzed by ELISA for BSA- or SRBC-specific IgG as previously described (30, 32). A BSA-specific standard serum, affinity-purified on BSA-Sepharose, was used to determine the concentrations of BSA-specific IgG. Standard curves and calculations were performed using a Softmax program (Molecular Devices, Menlo Park, CA). To determine the total numbers of B cells producing SRBC-specific IgM, a modified version of the Jerne hemolytic PFC assay was used (33).

Statistical differences between the control and the experimental groups were determined by Student’s t test.

IgM is known to enhance Ab responses to particulate Ags, such as erythrocytes and malaria parasites, whereas enhancement of responses to soluble protein Ags has been shown only occasionally, using KLH as Ag (17, 34). It may be that IgM needs to bind to a large Ag to take on the configurational change necessary for its ability to activate C, which is known to be required for the enhancing effect (24). For these reasons IgM-mediated enhancement was here studied in the classical SRBC system. This system also allows detection of single Ab-producing B cells using the sensitive hemolytic PFC assay (35).

CR1/2−/− and CR1/2+/+ mice were injected with 2 × 105 SRBC with or without IgM anti-SRBC. All groups were also given HRBC as a specificity control. IgM increased the IgG anti-SRBC response in CR1/2+/+ mice (Fig. 1,a), but not in CR1/2−/− mice (Fig. 1,b). There was no enhancement of the response to HRBC (data not shown). To determine whether the nonresponsiveness is also present at the single-cell level, CR1/2−/− and CR1/2+/+ mice were challenged as described above, and the numbers of IgM anti-SRBC-producing spleen cells were analyzed. No enhancement was detected in CR1/2−/− mice, whereas IgM induced a 4-fold enhancement of the direct PFC response in CR1/2+/+ mice (Table I, Expt. 1). Using an alternative approach, normal BALB/c mice were treated with 200 μg of anti-CR1/2 mAbs (7G6) 24 h before challenge with SRBC and IgM, a protocol shown previously to inhibit Ab responses to erythrocytes (5). The results show that IgM was unable to enhance Ab responses after in vivo blocking of CR1/2 (Table I, Expt. 2). Again, no enhancement of the HRBC-specific response was seen.

FIGURE 1.

Lack of IgM-mediated enhancement in CR1/2−/− mice. Groups of four or five CR1/2−/− or CR1/2+/+ mice were immunized with 2 × 105 SRBC and 4 × 105 HRBC with or without IgM anti-SRBC (0.1 ml; hemagglutination titer, 1:16). On the indicated days the IgG anti-SRBC and anti-HRBC titers were measured by ELISA. All sera were tested at a dilution of 1:10. The data shown are representative of three independent experiments. ∗, p < 0.05; ∗∗∗, p < 0.001.

FIGURE 1.

Lack of IgM-mediated enhancement in CR1/2−/− mice. Groups of four or five CR1/2−/− or CR1/2+/+ mice were immunized with 2 × 105 SRBC and 4 × 105 HRBC with or without IgM anti-SRBC (0.1 ml; hemagglutination titer, 1:16). On the indicated days the IgG anti-SRBC and anti-HRBC titers were measured by ELISA. All sera were tested at a dilution of 1:10. The data shown are representative of three independent experiments. ∗, p < 0.05; ∗∗∗, p < 0.001.

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Table I.

CRs and IgM in B cell responses to SRBCa

Expt.MiceImmunizationPFC Anti-SRBCb (log10 ± SD)pcPFC Anti-HRBCb (log10 ± SD)pc
CR1/2+/+ 2 × 105 SRBC+ 4× 105 HRBC 3.37 ± 0.48 3.51 ± 0.39 
   (2,334)  (3,220)  
 CR1/2+/+ IgM+ 2× 105 SRBC+ 4× 105 HRBC 4.02 ± 0.20 <0.01 3.50 ± 0.47 NS 
   (10,372)  (3,156)  
 CR1/2−/− 2× 105 SRBC+ 4× 105 HRBC 1.99 ± 0.41 2.68 ± 0.28 
   (99)  (476)  
 CR1/2−/− IgM+ 2× 105 SRBC+ 4 × 105 HRBC 1.91 ± 0.21 NS 2.76 ± 0.34 NS 
   (82)  (581)  
BALB/c 3× 105 SRBC+ 3× 105 HRBC 2.60 ± 0.42 4.43 ± 0.42 
   (398)  (26,915)  
 BALB/c IgM+ 3× 105 SRBC+ 3× 105 HRBC 4.30 ± 0.15 <0.001 4.69 ± 0.13 NS 
   (19,953)  (48,978)  
 BALB/c+ 7G6 3× 105 SRBC+ 3× 105 HRBC 1.99 ± 0.35 2.03 ± 0.41 
   (98)  (107)  
 BALB/c+ 7G6 IgM+ 3× 105 SRBC+ 3× 105 HRBC 1.70 ± 0.0 <0.001 2.36 ± 0.27 NS 
   (50)  (229)  
CR1/2−/− 1× 108 SRBC 3.21 ± 0.30   
   (1,622)    
 CR1/2+/+ 1× 108 SRBC 4.01 ± 0.33 <0.005   
   (10,232)    
 CR1/2−/− 2× 105 SRBC 2.23 ± 0.32   
   (170)    
 CR1/2+/+ 2× 105 SRBC 3.56 ± 0.67 <0.005   
   (3,630)    
Expt.MiceImmunizationPFC Anti-SRBCb (log10 ± SD)pcPFC Anti-HRBCb (log10 ± SD)pc
CR1/2+/+ 2 × 105 SRBC+ 4× 105 HRBC 3.37 ± 0.48 3.51 ± 0.39 
   (2,334)  (3,220)  
 CR1/2+/+ IgM+ 2× 105 SRBC+ 4× 105 HRBC 4.02 ± 0.20 <0.01 3.50 ± 0.47 NS 
   (10,372)  (3,156)  
 CR1/2−/− 2× 105 SRBC+ 4× 105 HRBC 1.99 ± 0.41 2.68 ± 0.28 
   (99)  (476)  
 CR1/2−/− IgM+ 2× 105 SRBC+ 4 × 105 HRBC 1.91 ± 0.21 NS 2.76 ± 0.34 NS 
   (82)  (581)  
BALB/c 3× 105 SRBC+ 3× 105 HRBC 2.60 ± 0.42 4.43 ± 0.42 
   (398)  (26,915)  
 BALB/c IgM+ 3× 105 SRBC+ 3× 105 HRBC 4.30 ± 0.15 <0.001 4.69 ± 0.13 NS 
   (19,953)  (48,978)  
 BALB/c+ 7G6 3× 105 SRBC+ 3× 105 HRBC 1.99 ± 0.35 2.03 ± 0.41 
   (98)  (107)  
 BALB/c+ 7G6 IgM+ 3× 105 SRBC+ 3× 105 HRBC 1.70 ± 0.0 <0.001 2.36 ± 0.27 NS 
   (50)  (229)  
CR1/2−/− 1× 108 SRBC 3.21 ± 0.30   
   (1,622)    
 CR1/2+/+ 1× 108 SRBC 4.01 ± 0.33 <0.005   
   (10,232)    
 CR1/2−/− 2× 105 SRBC 2.23 ± 0.32   
   (170)    
 CR1/2+/+ 2× 105 SRBC 3.56 ± 0.67 <0.005   
   (3,630)    
a

In Expt. 1, CR1/2−/− and CR1/2+/+ mice (n = 5–7) were immunized i.v. with indicated amounts of SRBC and HRBC with or without IgM anti-SRBC (hemagglutination titer, 1:16). In Expt. 2, BALB/c mice (n = 4–5), untreated or pretreated with 20 μg anti-CR1/2 mAb 7G6 24 h earlier, were immunized i.v. with indicated amounts of SRBC and HRBC with or without IgM anti-SRBC (hemagglutination titer, 1:16). In Expt. 3, CR1/2−/− and CR1/2+/+ mice (n = 5) were immunized i.v. with the indicated amounts of SRBC.

b

Direct PFC numbers expressed as log10 PFC/spleen ± SD were determined 6 days after immunization. Numbers in parentheses are the geometrical means.

c

Experimental value vs control value (c) as determined by Student’s t test; NS (p > 0.05).

These experiments were based on the assumption that enhancement by IgM would be detectable in CR1/2−/− mice, i.e., that the mice are responsive to SRBC under some conditions. To test this, we studied the Ab response after immunization with a high dose of SRBC (1 × 108/mouse). The numbers of SRBC-specific direct PFC were determined 3, 6, and 9 days after challenge. CR1/2−/− mice produced fewer SRBC-specific B cells than CR1/2+/+ mice at both high and low doses of SRBC challenge at all days tested (Table I, Expt. 3; only results from day 6 shown). The response in CR1/2−/− animals, however, rose from 170 to 1622 PFC/spleen when the high dose of Ag was used, demonstrating that these mice are able to produce Ab in response to high dose immunization. Therefore, the lack of IgM-mediated enhancement is most likely not due to an overall inability to respond to SRBC, but to a selective block in the IgM/C-dependent pathway of B cell activation.

IgG and IgE have dual immunoregulatory capacity. Whereas administration together with soluble Ags results in enhancement, administration with particulate Ags usually causes specific suppression of Ab responses (33, 34, 36). Therefore, to study IgE- and IgG-mediated enhancement, soluble protein Ags were used. CR1/2−/− and CR1/2+/+ mice were challenged with 20 μg of BSA-TNP alone or in complex with 50 μg of IgE or IgG2a anti-TNP mAbs. All animals received 20 μg of OVA as a specificity control. Sera were analyzed by ELISA 7–28 days after priming. Both strains responded to challenge with IgG2a/BSA-TNP (Fig. 2, a and b) or IgE/BSA-TNP (Fig. 2, c and d). The magnitude of the response to IgG2a/Ag was slightly lower in CR1/2−/− mice, but the difference compared with CR1/2+/+ mice was not significant. No enhancement of the IgG anti-OVA responses was detected (data not shown).

FIGURE 2.

Normal IgG2a- and IgE-mediated enhancement in CR1/2−/− mice. Groups of five CR1/2+/+ (left) or CR1/2−/− (right) mice were injected with 20 μg of BSA-TNP alone or in complex with 50 μg of IgG2a anti-TNP (a and b) or 50 μg of IgE anti-TNP (c and d). OVA (20 μg) was given to all mice as a specificity control. On the indicated days mice were bled, and the IgG anti-BSA and anti-OVA (data not shown) titers were measured by ELISA. Data are representative of three independent experiments. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

FIGURE 2.

Normal IgG2a- and IgE-mediated enhancement in CR1/2−/− mice. Groups of five CR1/2+/+ (left) or CR1/2−/− (right) mice were injected with 20 μg of BSA-TNP alone or in complex with 50 μg of IgG2a anti-TNP (a and b) or 50 μg of IgE anti-TNP (c and d). OVA (20 μg) was given to all mice as a specificity control. On the indicated days mice were bled, and the IgG anti-BSA and anti-OVA (data not shown) titers were measured by ELISA. Data are representative of three independent experiments. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

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To determine whether CR1/2−/− mice are able to respond to Ag administered with adjuvant, CR1/2−/− and CR1/2+/+ mice were challenged s.c. with various amounts of BSA in CFA or alum. Fig. 3 shows that both CR1/2−/− and CR1/2+/+ mice were able to produce high titers of BSA-specific IgG. The response to 2 μg was lower in CR1/2−/− than in CR1/2+/+ mice, but the difference was not significant.

FIGURE 3.

Ab responses in CR1/2−/− mice immunized with Ag in CFA or alum. Groups of five CR1/2+/+ (left) or CR1/2−/− (right) mice were immunized with 2, 20, or 200 μg of BSA in alum (a and b) or CFA (c and d). On the indicated days mice were bled, and the IgG anti-BSA titers were measured by ELISA. NMS, normal mouse serum.

FIGURE 3.

Ab responses in CR1/2−/− mice immunized with Ag in CFA or alum. Groups of five CR1/2+/+ (left) or CR1/2−/− (right) mice were immunized with 2, 20, or 200 μg of BSA in alum (a and b) or CFA (c and d). On the indicated days mice were bled, and the IgG anti-BSA titers were measured by ELISA. NMS, normal mouse serum.

Close modal

CR1/2−/− mice were able to produce high Ab titers after immunization with soluble Ag complexed to IgE or IgG2a. This shows that the requirement for CR1/2 in Ab responses is not absolute, and, moreover, that responses can be initiated without using conventional adjuvants. IgE-mediated enhancement is known to require CD23 (20, 21). Despite the inability of IgE to activate C, a possible link to the C system exists, since human CD23 has been described as a ligand for CR2 (37). The ability of IgE to enhance in CR1/2−/− mice, however, shows that the enhancement of Ab responses in mice, although mediated by CD23, does not require CR2. Whether IgG-mediated enhancement requires C activation has been a controversial issue. Early studies showed that CVF treatment to deplete C abolished IgG-mediated enhancement of memory B cells (38). Furthermore, a correlation between the ability of a panel of IgG mAbs to activate C and their enhancing effects suggested that C activation was involved (17, 36). However, mutant IgG2a and normal IgG1 mAbs, both unable to activate the classical pathway, were subsequently shown to be potent up-regulators of Ab responses (18). Moreover, enhancement by normal IgG2a and IgG1 was severely diminished in mice lacking the FcR γ-chain (19). The FcR γ-chain is associated with FcγRI and FcγRIII, but has not been shown to affect the C system. Therefore, the results suggested that FcγRs are of primary importance in IgG-mediated enhancement. The ability of IgG to up-regulate Ab responses without CR1/2, demonstrated in the present report, strengthens this conclusion. The exact molecular mechanisms by which IgE and IgG up-regulate Ab responses are not understood. Efficient uptake and presentation of Ag in the form of IgE or IgG complexes to T cells is known to take place in vitro (39, 40, 41, 42, 43, 44) and may also be the general mechanism in vivo. If this is indeed the case, it suggests that when efficient T cell help is available, signaling through the BCR, without costimulation via the CR2/CD19/TAPA-1 complex, is sufficient to activate B cells. Because IgG and IgE are more abundant in secondary than in primary Ab responses, it may be that secondary Ab responses are less C dependent than primary Ab responses, which is in line with previous experimental data (7, 8).

Adjuvants have classically been used to augment immune responses, but their mechanism of action is unclear. We show here that mice lacking CR1/2 are able to respond to T-dependent Ags administered in adjuvants. These results agree with previous data showing that CVF-treated animals respond to Ag administered in adjuvant (11, 45) and are another example of CR1/2-independent induction of humoral responses.

We also confirm previous observations that CR1/2−/− mice have severely impaired serum Ab responses to SRBC alone (7) and extend them to show that this is paralleled by fewer Ag-specific B cells in the spleens of CR1/2−/− mice after Ag challenge. This finding is compatible with the observations of smaller and fewer GCs in CR1/2−/− mice following ΦX174 challenge (8).

Previous findings that IgM, Ag, and C work together to enhance Ab responses (24) and that normal Ab responses require CR1/2 (5, 7, 8, 9) implied that IgM-mediated enhancement operates via CR1/2. We here provide the first experimental evidence that this is indeed the case. We have used preformed Ag-specific IgM, but in a physiological, primary response Ag would initially form complexes with natural IgM. The importance of natural IgM in the induction of immunity is supported by recent observations. Gene-targeted mice, lacking secretory IgM but able to express surface IgM and to secrete IgG and IgA, had diminished Ab responses after immunization with KLH-4-hydroxy-3-nitrophenyl (46, 47). Responses were reconstituted when IgM from normal mouse serum was administered to targeted mice before challenge (46). Therefore, it appears likely that Ab responses to low doses of Ag involves recognition of the Ag by natural IgM followed by C activation and ligation of CR1/2, which would lead to production of early specific IgM and, in turn, further enhance the positive feedback loop.

The precise mechanism behind the role of CR1/2 in Ab responses is not known. Positive signaling to Ag-specific B cells via IgM/Ag/C coligated to the CR2/CD19/TAPA-1 complex may be the signal necessary to break threshold responses to low dose Ags (48, 49). CR1/2-facilitated Ag presentation to T cells by opsonization of immune complexes has been seen using in vitro systems (50, 51, 52), although T cell priming in vivo can occur without functional CR1/2 (53) or without the presence of C3 (13). CR1/2 also affect B cell retention/survival within lymphoid follicles and GCs (54). Regardless of which molecular mechanism(s) is involved, the data presented here reveal two important aspects of the in vivo role of CR1/2. First, CR1/2 is a crucial receptor in triggering responses to IgM/Ag complexes, which is probably the first step in Ab production. Second, the need for CR1/2 can be circumvented when Ags are administered in complex with IgG2a or IgE, isotypes typical of secondary responses, or together with adjuvants, a system mimicking natural inflammatory responses.

We thank I. Brogren for excellent technical assistance, Drs. M. Wabl, P. Coulie, and T. Kinoshita for hybridoma cell lines, and Dr. B. Ersson for purification of IgM.

1

This work was supported by the Swedish Medical Research Council; the Swedish Foundation for Health Care Sciences and Allergy Research; King Gustaf V’s 80 Year Foundation; Ellen, Walter, and Lennart Hesselman’s Foundation; Hans von Kantzow’s Foundation; and The Swedish Institute.

5

Abbreviations used in this paper: CFV, cobra venom factor; CR, complement receptor; BCR, B cell receptor; TNP, 2,4,6-trinitrophenyl; HRBC, horse erythrocytes; PFC, hemolytic plaque-forming cells; GC, germinal center; KLH, keyhole limpet hemocyanin; FcγR, Fc receptor for IgG.

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