Complement receptor type 2 (CR2/CD21), in association with CD19, plays an important role in enhancing mature B cell responses to opsonized Ags. We have shown that mice expressing a human CR2/CD21 (hCR2/CD21) transgene during the CD43+/CD25 late pro-B cell stage of B cell development demonstrate marked changes in subsequent B cell ontogeny. In the present study, we show that the humoral immune response to the T cell-dependent Ag, sheep RBC, is muted severely in a manner inversely proportional to B cell expression level of hCR2. Individual Ag-specific IgG isotypes vary in the degree to which they are affected but all are reduced while IgM titers are normal. A substantial reduction in germinal centers, both in size and frequency, in the spleens of immunized hCR2 transgenic mice demonstrates a failure to maintain germinal center reaction. However, both IgM expression levels and LPS-proliferative responses appear fully intact in B cells from hCR2-positive mice, suggesting that this alteration in B cell phenotype is different qualitatively from that of specific Ag-defined anergy models. These data suggest that the unresponsiveness to T-dependent Ags displayed by hCR2-positive B cells is linked to an increase in the level of stimulus required to propel the B cell into a fully activated state and thus a normal humoral immune response to Ags. We conclude that this phenotype and these mice may offer an additional means to dissect mechanisms underlying B cell tolerance and Ag responsiveness both in bone marrow and periphery.

The production of immature B cells in the bone marrow is estimated to be in the order of 10–20 million/day. Of these, only 10–20% reach the periphery, and as few as 3% contribute to the mature B cell pool (1, 2, 3, 4). This illustrates the fundamental mechanism that has evolved to regulate the generation of autoreactive B cells, namely negative selection through apoptosis. According to the clonal selection hypothesis, B cells that bind too strongly to self-Ags as they develop in the bone marrow will undergo apoptosis (5, 6, 7). However, the exact criteria which govern the elimination of self-reactive B cells as they develop are not yet known. Identifying these factors will be crucial to understanding the mechanisms that lie behind the breakdown of tolerance and the generation of autoimmune syndromes.

Deletion of self-reactive B cells is not the only mechanism whereby the immune system can regulate autoreactive B cells. At least two other mechanisms, receptor editing and anergy, have been shown to limit the generation of self-reactive Abs during B cell development in the bone marrow (8, 9, 10, 11, 12). Anergy or B cell unresponsiveness to Ags represents a means of tolerizing immature B cells that have left the bone marrow and entered the periphery. In this way, anergy may represent the last line of defense against immature B cells becoming self-reactive mature B cells and setting up an autoimmune response. The criteria that govern the decision of a B cell to become anergic or to become activated are still not fully understood. Anergy itself appears to be graded into various levels of unresponsiveness according to the situation that drives the B cell down that path. For example, Goodnow’s elegant studies using mice that have B cells with transgenic (tg) 3 BCR directed against hen egg lysozyme (HEL) and that also produce soluble HEL (sHEL) in the periphery illustrate one of the most extreme forms of anergy (10, 13). In this model, the B cells demonstrated a markedly reduced surface IgM expression and were found to be short lived due to their exclusion from B cell follicles. In contrast, the anti-ssDNA B cells described by Erikson and colleagues (14, 15) probably represent a far milder form of anergy. In that study, B cells do not down-regulate surface IgM, are long-lived, and can occupy B cell follicles. Examination of signal transduction potential demonstrates that the anergic B cells were still partially functional, contrasting the almost complete loss of signal transduction noted in anergic B cells from the anti-HEL/sHEL model (14, 15, 16). Notably, it appears that all anergic B cells share an impaired ability to differentiate to Ab-secreting plasma cells in response to Ag and LPS treatment, yet anergy has been shown to be reversible, and thus, all anergic B cells have to a greater or lesser degree an ability to provide the source of autoreactive Ab that may ignite autoimmune disease (15, 17).

Expression of complement receptor type 2 (CR2) on human B lymphocytes was first studied by Tedder et al. (18), who found that CR2 was not detectable on pre-B or immature B cells or late-stage plasma cells but was easily detectable on mature B cells. Mouse CR2 (mCR2) was also found to display this tightly regulated expression pattern (19). Human CR2 (hCR2) and mCR2 are also very similar in many other respects because they share ∼67% homology at the nucleotide level and 58% homology in the protein sequence (20). Both exhibit a molecular mass of ∼140–150 kDa, and hCR2 binds mouse and human C3d with similar affinities (21). The importance of CR2 in promoting B cell activation to Ags coated with C3d was demonstrated clearly by Dempsey et al. (22), who found that when mice were immunized with Ags coupled to C3d, up to a 10,000-fold lower dose of coupled Ags, compared with uncoupled Ags, was required to generate a detectable Ab response. Importantly, cross-linking of CR2 in conjunction with the BCR has also been shown to rescue resting splenic B cells from apoptosis (23). These findings clearly indicated that binding of a C3d-coated immune complex through BCR and CR2 could provide the level of signal required to rescue B cells from elimination and therefore may also provide sufficient signal to reverse anergy in the periphery.

Previously, we created mice expressing hCR2 cDNA under the control of a B cell-specific λ promoter/enhancer minigene to investigate the role of CR2 in B cell fate and to create a model to investigate the use of human C3d-tagged vaccines in vivo (24). We found a 60% reduction in B cell numbers in the periphery of mice that expressed the highest levels of hCR2 protein irrespective of whether the mice did or did not express endogenous mCR2. These mice also displayed a reduction in the level of serum IgG and a marked reduction in production of specific Ig in response to the T-dependent Ag 4-hydroxy-3-nitrophenyl-keyhole limpet hemocyanin (NP-KLH) (24). These defects were all related primarily to the premature expression of CR2 on the B cell surface during the bone marrow-associated phase of B cell development. However, the limited analysis of the immune response in the previous study left several key questions regarding the effect of hCR2 transgene on B cell function unanswered. First, in the absence of endogenous mCR1/2, what effect does the level of hCR2 expression on the mouse B cell have on the immune response? Second, what effect does hCR2 expression have on production of the various IgG isotypes and the formation of a stable germinal center (GC) response? Third, does the premature expression of CR2 on the B cell alter the way it perceives Ag? To investigate these questions, we conducted detailed analysis of B cell activation markers and immune response to a second T-dependent Ag, SRBC, in three lines of hCR2-tg mice with differing hCR2 protein expression in the absence of endogenous mCR2.

Blood was collected into heparin following a tail vein nick, and cells were pelleted and washed once in cold PBS. Bone marrow B cells were collected by flushing mouse femurs with cold PBS. Splenocytes were isolated from whole spleens by disrupting the spleen between two frosted glass slides in PBS buffer and transferred to 15 ml of conical tubes on ice. Large debris settled after a 10-min incubation, and the supernatant was transferred to a new tube. Cells were pelleted by centrifugation and washed once with staining buffer (PBS, 1% FCS, and 0.02% sodium azide). Samples were incubated with 0.5–1 ml of RBC lysis buffer (0.83% NH4Cl, 0.1% KCO3, and 0.1 mM EDTA) and incubated at room temperature for 1–2 min. The cells were then washed with 1 ml of staining buffer. Cells were counted, and 1–3 × 106 cells/ml were used per analysis. Cells were stained as described below.

Purified and biotin conjugated mAb 171 (anti-hCR2, IgG1 isotype) (25), IgG1 isotype control, and the polyclonal anti-hCR2 Ab were produced in the laboratory following standard methods. 2.4G2 (anti-mCD16/mCD32, Fc block), PE-conjugated B-Ly-4 (anti-hCR2) and CD19, allophycocyanin- or FITC-conjugated RA3-6B2 (anti-mCD45R, B220), FITC-conjugated anti-B7.1 (CD80), FITC-conjugated anti-B7.2 (CD86), FITC-conjugated Gl-7, biotin-conjugated anti-CD95 (jo-1), biotin-conjugated anti-I-Ab (MHC II), biotin-conjugated anti-CD44, biotin-conjugated anti-CD25 (Il-2Rα-chain), biotin-conjugated anti-CD69, and streptavidin (SA)-allophycocyanin were all obtained from BD Pharmingen. SA-FITC and SA-PE were obtained from Jackson ImmunoResearch Laboratories (Stratech). FITC-conjugated anti-IgM, purified goat anti-mouse IgG/IgM, and alkaline phosphatase-conjugated goat anti-mouse Ig isotype secondaries were obtained from Caltag Laboratories.

The generation and analysis of mice expressing high (hCR2high), intermediate (hCR2int), or low levels (hCR2low) of hCR2 were described previously (24). To screen for hCR2-expressing pups, DNA was extracted from an ear punch biopsy to allow PCR analysis. The ear biopsies were incubated with detergent lysis buffer (10 mM Tris-HCl, 20 mM KCl, 5% NP40 v/v, and 5% Tween 20 v/v (pH 8.0)) for 15 min at 95°C, the samples were cooled, and 1 μg/ml proteinase K was added. Samples were incubated overnight at 56°C, followed by an incubation at 95°C for 20 min. PCR analysis was conducted on 3 μl of detergent lysis supernatant. Confirmed founders were backcrossed subsequently onto the Cr2−/− background and followed by PCR using two independent primer sets (5′-GTGAATTAAGGCCTGGACTTCACTT-3′ and 5′-TTATCCCAGGTTCCATCCAC-3′; 5′-ATGAGGGGCAGGTTGGCTCC-3′ and 5′-GGCAGGAAAACTTTCTATATGG-3′) at cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. Both PCRs were conducted for 27 cycles. Genotyping was also confirmed by flow cytometric analysis. All experiments reported herein were conducted using hCR2+ or hCR2 littermates on F6-9 backcross to the Cr2−/− background (26) with age-matched C57BL/6 mice, purchased from Harlan Sprague Dawley, acting as wild-type (Cr2+/+ hCR2) control mice where appropriate. All animal experiments were conducted with permission of the local ethical review and in strict accordance with Home Office guidelines.

Cells were resuspended in staining buffer (1× PBS, 1% BSA, and 0.02% sodium azide) containing 10 μg/ml 2.4G2 Ab to block FcRs. After a 15-min incubation on ice, cells were washed in staining buffer. Cells were resuspended in 100 μl of staining buffer containing the appropriate test Ab (used at between 1/50–1/400) and 1 μl of anti-B220-FITC (to label mouse B cells). Cells were incubated for 30 min on ice in the dark. After incubation, cells were washed in staining buffer two to three times and then incubated with the appropriate secondary Ab (used at 1/1000 in staining buffer). Following incubation, cells were washed as above and then resuspended in 1% paraformaldehyde. Flow cytometry was conducted using a BD FACSCalibur.

SRBCs (TCS Bioscience) were washed three times with PBS, and a suspension was made containing 1 × 108 SRBC/ml. Mice were injected i.p. at days 0 and 28 with 500 μl of SRBC suspension. Serum was collected from tail vein bleed at days 14, 21, 28, and 36. Detection of Ab to SRBC was conducted by ELISA essentially as previously described by Heyman et al. (27). To calculate relative units, the mean OD at 405 nm from triplicate wells was compared with a semilog standard curve of OD measurements (x) vs titrated standard serum.

Mice were injected with SRBC by the i.p. route as described above, and spleens were collected on day 8. Spleens were divided such that ∼75% was mounted in OCT compound (EMS Laboratories) and snap frozen in isopentane for immunohistochemistry, and 25% was used for flow cytometric analysis in the manner outlined above. Frozen sections (10 μm) were cut on a CME cryostat (Thermo Shandon) and dried at 37°C for 30 min before being fixed for 10 min in acetone. After a brief wash in PBS, endogenous peroxidase activity was quenched by incubating the section with 0.1% H2O2 in PBS for 20 min. Biotin and avidin binding sites were then blocked using a biotin/avidin blocking kit (Vector Laboratories). Sections were incubated with 1:100 rat anti-mouse IgD (BD Pharmingen) and 1:300 biotin-conjugated PNA (Vector Laboratories) in 1% BSA/PBS for 1 h at room temperature. Slides were washed three times with PBS. Sections were then incubated with 1:300 rabbit anti-rat HRP (Stratech) and 1:400 Extra-Avidin-AP (Sigma-Aldrich) for 1 h at room temperature. Slides were washed three times in PBS. The sections were then developed using a BCIP/NBT alkaline phosphatase kit (Vector Laboratories), according to the manufacturer’s instructions, and after a brief wash in PBS, HRP was developed using 0.05% diaminobenzidine/0.02% H2O2. Slides were rinsed with tap water after development, dehydrated, and mounted using Surgipath (Bretton) mounting medium. Sections were viewed using a Leica microscope with attached digital camera and analyzed using Improvision Openlab software (Improvision).

We first analyzed the immune response to the T-dependent Ag SRBCs in Cr2−/− mice expressing hCR2 at different levels to evaluate the effect of hCR2 expression level on B cell responsiveness to Ags as measured by immune titer. Three lines of mice were investigated, which expressed hCR2 on the B cell surface in the absence of endogenous mCR1/CR2 at levels equivalent to 25–33% (hCR2high), 15% (hCR2int), and 1–2% (hCR2low) that seen on a normal human B cell. First, we found that, as previously reported (26), there is a significant reduction in the anti-SRBC IgG humoral immune response in Cr2−/− mice as compared with Cr2+/+ wild-type mice (Fig. 1, c–f). Furthermore, all hCR2+ mice examined displayed a reduction in IgG isotypes titers in excess of their Cr2−/−hCR2 littermates, with the magnitude of the reduction correlating with the level of hCR2 expression (Fig. 1, c–f). Analysis of individual IgG isotypes revealed that all were similarly affected; the largest change was seen with IgG2b (Fig. 1,e). The presence of hCR2 on the mouse B cells affected the generation of IgG1 and IgG2b isotypes the most at day 21 of the immune response, but IgG2a and IgG3 were reduced significantly in the hCR2high line. However, in all hCR2-expressing lines, we found that IgM and IgA serum titers in response to SRBC injection were similar when compared with hCR2 littermates (Fig. 1, a and b). After a second SRBC injection at day 28 and serum collection at day 35, titers were examined again. IgM titers were slightly lower and IgA titers remained relatively unchanged after the boost in all animals examined (Fig. 2, a and b). Although a similar fold increase in IgG titers to SRBC boost was found in hCR2+ mice when compared with hCR2 mice (Fig. 2, c–f), IgG titers still remained significantly lower than hCR2 littermates. In particular, a significant reduction in the IgG2a isotype was seen in all hCR2+ lines at day 36 when compared with hCR2 littermates at this time. Furthermore, all hCR2+ mice still showed a distinct correlation between increased hCR2 expression level and reduced response to SRBCs.

FIGURE 1.

IgG isotype immune titers are reduced in response to SRBCs at day 21. Mice were injected with 5 × 108 SRBCs at day 0 and bled on day 21. To determine the levels of Ab to SRBC, individual mouse sera were analyzed in quadruplicate, and the average relative units plotted above. Age-matched mouse groups include Hi (hCR2high/mCR1/2−/−), Int (hCR2int/Cr2−/−), Lo (hCR2low/Cr2−/−), Neg (hCR2/Cr2−/−), and WT (wild-type Cr2+/+ C57BL/6); each circle represents one animal, and the bar represents the mean response in each group. Student’s t test was used to determine the p values. ∗∗, p < 0.005; ∗, p < 0.05 when compared with Neg.

FIGURE 1.

IgG isotype immune titers are reduced in response to SRBCs at day 21. Mice were injected with 5 × 108 SRBCs at day 0 and bled on day 21. To determine the levels of Ab to SRBC, individual mouse sera were analyzed in quadruplicate, and the average relative units plotted above. Age-matched mouse groups include Hi (hCR2high/mCR1/2−/−), Int (hCR2int/Cr2−/−), Lo (hCR2low/Cr2−/−), Neg (hCR2/Cr2−/−), and WT (wild-type Cr2+/+ C57BL/6); each circle represents one animal, and the bar represents the mean response in each group. Student’s t test was used to determine the p values. ∗∗, p < 0.005; ∗, p < 0.05 when compared with Neg.

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FIGURE 2.

The boost response to SRBCs remains intact in the hCR2-tg mice, but levels of IgG isotype titers remain lower than both wild-type or Cr2−/− mice. Mice were injected with 5 × 108 SRBCs at day 0 and bled on days 21 and 35. To determine the levels of Ab to SRBCs, individual mouse sera were analyzed in quadruplicate, and the average relative units plotted above. Age-matched mouse groups include Hi (♦ hCR2high/Cr2−/−), Int (▴ hCR2int/Cr2−/−), Lo (○ hCR2low/Cr2−/−), Neg (□ hCR2/Cr2−/−), and WT (▪ Cr2+/+ C57BL/6 wild type). Student’s t test was used to determine the p values.∗, p < 0.05; ∗∗, p < 0.01 when compared with Neg.

FIGURE 2.

The boost response to SRBCs remains intact in the hCR2-tg mice, but levels of IgG isotype titers remain lower than both wild-type or Cr2−/− mice. Mice were injected with 5 × 108 SRBCs at day 0 and bled on days 21 and 35. To determine the levels of Ab to SRBCs, individual mouse sera were analyzed in quadruplicate, and the average relative units plotted above. Age-matched mouse groups include Hi (♦ hCR2high/Cr2−/−), Int (▴ hCR2int/Cr2−/−), Lo (○ hCR2low/Cr2−/−), Neg (□ hCR2/Cr2−/−), and WT (▪ Cr2+/+ C57BL/6 wild type). Student’s t test was used to determine the p values.∗, p < 0.05; ∗∗, p < 0.01 when compared with Neg.

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The reduction in IgG titers observed in all lines of hCR2-expressing mice suggested to us that the GC response might be impaired. The general splenic architecture of unimmunized mice appeared unaltered in the hCR2-tg mice as compared with hCR2 littermates, and absolute B cell follicle numbers were found to be similar in all groups of mice analyzed (data not shown). However, wild-type mice immunized with SRBCs were found to possess large PNA-positive GCs in ∼66% of B cell follicles in examined sections of the spleen (Fig. 3, a and b), and in general, follicles were found to possess multiple GCs. GC number and size were found to be reduced in Cr2−/− mice as reported previously (Fig. 3,b; Refs. 28, 29, 30). Furthermore, expression of hCR2 in Cr2−/− mice led to a further reduction in both the frequency and numbers of GCs per follicle when compared with their hCR2 littermates (Fig. 3, a and b). To further quantitate the changes in GC B cell numbers, we used flow cytometry on splenocytes and GL-7 as marker for GC B cells (31, 32). We found a significant reduction in total splenic GC B cell numbers in hCR2high- and hCR2int-tg lines in comparison to the hCR2 littermates (Fig. 3, c and d). The hCR2low line also showed reduction in GL-7+ B cells that just failed to reach significance (Fig. 3, b and d). CD38 expression is also down-regulated on both GC B cells and mature plasma cells in the mouse (33). Therefore, to confirm the data obtained using GL-7, we also stained for CD38. In the hCR2high mice and, to a lesser extent, in both the hCR2int and hCR2low mice, we found a significant decrease in the number of B cells, which were CD38low when compared with hCR2 littermates, further supporting the conclusion that GC B cell numbers were reduced (Fig. 3 e).

FIGURE 3.

Analysis of hCR2-tg mouse spleens reveal a defective GC response. Mice were injected with 5 × 108 SRBCs, and tissues were collected from all mice as outlined in Materials and Methods at day 8 (a). Splenic sections Hi (hCR2high/Cr2−/−), Int (hCR2int/Cr2−/−), Lo (hCR2low/Cr2−/−), Neg (hCR2/Cr2−/−), and WT (Cr2+/+ C57BL/6 wild type) were stained with anti-IgD-HRP to label B cells (follicles), and peanut lectin (agglutinin)-alkaline phosphatase was used to label the GCs. The sections displayed are representative of the six discontinuous sections per spleen and greater than five animals per group that were analyzed. b, The number of GC-containing follicles vs total number of follicles present in the sections analyzed as described above. c, Flow cytometry was conducted on isolated splenocytes on day 8 after injection of SRBCs. Cells were stained with B220-allophycocyanin to label B cells, and GL-7-FITC was used to label cells associated with the GC response. Representative staining of hCR2high and hCR2 littermates are shown. d, Combined analysis of the percentage of B220+GL-7+ cells found in each group of animals. e, Analysis of CD38 expression on splenic B cells. The percentage of B cells that were determined as being B220+CD38low is shown. A minimum of six mice per group were used. ∗, p < 0.05 when compared with hCR2 as determined by Student’s t test.

FIGURE 3.

Analysis of hCR2-tg mouse spleens reveal a defective GC response. Mice were injected with 5 × 108 SRBCs, and tissues were collected from all mice as outlined in Materials and Methods at day 8 (a). Splenic sections Hi (hCR2high/Cr2−/−), Int (hCR2int/Cr2−/−), Lo (hCR2low/Cr2−/−), Neg (hCR2/Cr2−/−), and WT (Cr2+/+ C57BL/6 wild type) were stained with anti-IgD-HRP to label B cells (follicles), and peanut lectin (agglutinin)-alkaline phosphatase was used to label the GCs. The sections displayed are representative of the six discontinuous sections per spleen and greater than five animals per group that were analyzed. b, The number of GC-containing follicles vs total number of follicles present in the sections analyzed as described above. c, Flow cytometry was conducted on isolated splenocytes on day 8 after injection of SRBCs. Cells were stained with B220-allophycocyanin to label B cells, and GL-7-FITC was used to label cells associated with the GC response. Representative staining of hCR2high and hCR2 littermates are shown. d, Combined analysis of the percentage of B220+GL-7+ cells found in each group of animals. e, Analysis of CD38 expression on splenic B cells. The percentage of B cells that were determined as being B220+CD38low is shown. A minimum of six mice per group were used. ∗, p < 0.05 when compared with hCR2 as determined by Student’s t test.

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Our initial analysis of early B cell progenitors indicated that hCR2 expression was higher on these cells when compared with mature B cells in the bone marrow and appeared to coincide with a block in B cell development (24). On further analysis, we find that hCR2 expression levels are reduced by up to 50% as B cells mature from B220+CD43+ to B220+CD43 B cells in the bone marrow of hCR2high mice (Table I). Similarly, hCR2int mice also demonstrate a small but significant reduction in hCR2 expression levels at this development phase. However, expression levels of hCR2 on B cells from hCR2low mice remain essentially unchanged in the bone marrow. Both the hCR2high and hCR2int mice displayed a further significant reduction of hCR2 expression levels on B cells isolated from the spleen, but the hCR2 expression levels seen on peripheral blood derived B cells were equivalent to that of the CD43 B cells of the bone marrow. Interestingly, the hCR2low mice express higher levels of hCR2 on their splenic and peripheral blood lymphocytes when compared with B cells isolated from the bone marrow. No significant differences in CD19 and IgM expression levels were observed between hCR2-tg and hCR2 littermates during B cell maturation (data not shown).

Table I.

hCR2 expression is down-regulated on mouse B cells as they maturea

BMb CD43+BM CD43SpleenBlood
hCR2high 59.8 ± 2.2 29.8 ± 3.7∗∗ 24.4 ± 1.4∗∗ 31.7 ± 1.1 
hCR2int 23.3 ± 2.4 20.7 ± 1.7∗ 16.1 ± 0.7∗ 21.2 ± 1.5 
hCR2low 7.1 ± 0.7 6.6 ± 1.4 8.2 ± 0.5∗ 8.4 ± 0.1 
hCR2 4.6 ± 0.8 4.6 ± 0.3 4.4 ± 0.4 4.2 ± 0.1 
BMb CD43+BM CD43SpleenBlood
hCR2high 59.8 ± 2.2 29.8 ± 3.7∗∗ 24.4 ± 1.4∗∗ 31.7 ± 1.1 
hCR2int 23.3 ± 2.4 20.7 ± 1.7∗ 16.1 ± 0.7∗ 21.2 ± 1.5 
hCR2low 7.1 ± 0.7 6.6 ± 1.4 8.2 ± 0.5∗ 8.4 ± 0.1 
hCR2 4.6 ± 0.8 4.6 ± 0.3 4.4 ± 0.4 4.2 ± 0.1 
a

Splenocytes, bone marrow, and peripheral blood lymphocytes were collected using standard methods. Cells were counted, and flow cytometry was carried out on the lymphocytes as described in Materials and Methods. All rows show mean fluorsecence units on 10,000 B220+ B cells from each tissue and each group of mice. Three mice were used per group, and Student’s t test was used to establish p values compared with the adjacent tissue. ∗∗, p < 0.001, ∗, p < 0.05.

b

BM, Bone marrow.

Previous studies have shown that B cells can display an activated phenotype despite being unresponsive to specific Ags in models of anergy (34, 35). To investigate whether B cells expressing hCR2 adopt a similar phenotype, we examined a battery of activation markers on unstimulated B cells in the spleen and blood of hCR2-tg mice. We found that expression of hCR2 was associated with increased activation marker expression in a dose-dependent manner (Table II). MHC class II expression was consistently increased on B cells isolated from spleen and blood in the hCR2high and hCR2int lines. CD44 was also markedly up-regulated on B cells isolated from the spleen and blood of hCR2high mice. Blood-derived B cells from hCR2int mice also demonstrated a clear increase in CD44 expression levels. In contrast, B cells from the hCR2low line exhibited a small but significant decrease in MHC II and CD44 expression levels in both compartments (Table II). The percentage of B cells expressing the activation markers B7.1 or B7.2 was also increased in splenic and blood-derived hCR2high B cells. A similar trend was seen in the hCR2int mice, but these increases failed to reach significance. Increased CD95/FAS expression on B cells has also been noted in certain forms of anergy (36, 37, 38). To examine whether hCR2-expressing B cells also exhibited this phenotype, CD95 expression levels were measured. The percentage of CD95-expressing B cells in hCR2high mice was increased significantly in the blood derived B cells and, although not significantly increased in the spleen, did show a trend to higher expression when compared with hCR2-tg mice (Table II).

Table II.

Activation markers are up-regulated on unstimulated B cells from hCR2 transgenic micea

PBLSplenocytes
hCR2highhCR2inthCR2lowNegativehCR2highhCR2inthCR2lowNegative
MHC II 80.7 ± 12.0∗∗∗ 70.8 ± 7.1∗∗ 50.6 ± 5.1 56.4 ± 9.1 110.5 ± 15.2∗∗ 106.7 ± 9.9∗∗ 82.8 ± 7.7∗ 91.9 ± 6.9 
CD44 209.3 ± 34.4∗ 219.0 ± 22.2∗ 160.7 ± 17.5∗ 184.8 ± 21.0 166.9 ± 22.3∗ 153.5 ± 18.8 158.4 ± 10.5 148.9 ± 20.0 
B7.1 11.1 ± 5.5∗ 9.2 ± 1.7 6.5 ± 1.1 7.9 ± 1.7 9.8 ± 3.0∗ 9.7 ± 3.6 6.8 ± 1.6 7.5 ± 1.5 
B7.2 2.7 ± 2.4∗ 0.9 ± 0.5 0.9 ± 0.3 1.3 ± 0.9 2.7 ± 1.4 2.9 ± 1.1 2.0 ± 0.4 2.3 ± 0.7 
CD95 6.4 ± 2.1∗ 5.2 ± 3.8 3.2 ± 0.7 3.6 ± 0.8 14.1 ± 4.4 14.0 ± 3.2 12.5 ± 2.7 12.7 ± 3.7 
PBLSplenocytes
hCR2highhCR2inthCR2lowNegativehCR2highhCR2inthCR2lowNegative
MHC II 80.7 ± 12.0∗∗∗ 70.8 ± 7.1∗∗ 50.6 ± 5.1 56.4 ± 9.1 110.5 ± 15.2∗∗ 106.7 ± 9.9∗∗ 82.8 ± 7.7∗ 91.9 ± 6.9 
CD44 209.3 ± 34.4∗ 219.0 ± 22.2∗ 160.7 ± 17.5∗ 184.8 ± 21.0 166.9 ± 22.3∗ 153.5 ± 18.8 158.4 ± 10.5 148.9 ± 20.0 
B7.1 11.1 ± 5.5∗ 9.2 ± 1.7 6.5 ± 1.1 7.9 ± 1.7 9.8 ± 3.0∗ 9.7 ± 3.6 6.8 ± 1.6 7.5 ± 1.5 
B7.2 2.7 ± 2.4∗ 0.9 ± 0.5 0.9 ± 0.3 1.3 ± 0.9 2.7 ± 1.4 2.9 ± 1.1 2.0 ± 0.4 2.3 ± 0.7 
CD95 6.4 ± 2.1∗ 5.2 ± 3.8 3.2 ± 0.7 3.6 ± 0.8 14.1 ± 4.4 14.0 ± 3.2 12.5 ± 2.7 12.7 ± 3.7 
a

Flow cytometry was carried out on PBL and splenocytes as described in Materials and Methods. The top two rows show mean fluorescence units on B220+ B cells from each marker and each group of mice. The lower three rows show the percentage of cells with above background levels of expression of each marker on B220+ B cells. At least six mice were used per group, and Student’s t test was used to establish p values. ∗∗∗, p < 0.0001, ∗∗, p < 0.001, ∗, p < 0.05 compared with negative.

Classical B cell anergy has been shown to produce B cells that are nonresponsive to LPS stimulation in culture (15, 17, 39, 40). To address whether the B cells that express hCR2 resemble anergic cells in this respect, splenic B cells isolated from hCR2high or hCR2 littermates were incubated with LPS in vitro for 72 h. B cell numbers were measured, and expression of B cell activation markers was assessed. Both hCR2-expressing and hCR2 littermate B cells proliferated to the same extent in culture, and the cell number increased 2.2- and 1.9-fold, respectively. Furthermore, polyclonal B cell activation, as measured by total levels of secreted Ab, was also found to be equivalent in both hCR2-expressing and hCR2 LPS-treated cultures (data not shown). LPS treatment increased all activation markers examined in both groups of mice, again suggesting the cells could be polyclonally activated to an equal degree (Table III). However, CD44 remained significantly higher on B cells isolated from the hCR2high mice after LPS treatment when compared with LPS-treated, hCR2 littermate controls. MHC II followed a similar pattern to CD44, but the difference in expression level between hCR2-expressing and hCR2 groups after LPS treatment was not found to be significant.

Table III.

Expression of activation markers on B cells from hCR2high or negative mice at 72 h after LPS stimulationa

MockLPS
NegativehCR2highNegativehCR2high
MHC II 76.3 ± 31.0 144.2 ± 25.2∗ 149.3 ± 16.2 160.5 ± 16.1 
CD44 185.8 ± 20.4 235.1 ± 20.1∗ 321.0 ± 15.0 427.6 ± 53.5∗ 
CD69 4.2 ± 2.6 2.28 ± 0.1 90.8 ± 12.5 115.5 ± 36.1 
CD25 7.3 ± 1.8 4.9 ± 0.3 62.8 ± 7.6 53.3 ± 1.1 
slgM 127.4 ± 11.8 114.2 ± 6.5∗ 205.7 ± 14.0 173.3 ± 24.2 
MockLPS
NegativehCR2highNegativehCR2high
MHC II 76.3 ± 31.0 144.2 ± 25.2∗ 149.3 ± 16.2 160.5 ± 16.1 
CD44 185.8 ± 20.4 235.1 ± 20.1∗ 321.0 ± 15.0 427.6 ± 53.5∗ 
CD69 4.2 ± 2.6 2.28 ± 0.1 90.8 ± 12.5 115.5 ± 36.1 
CD25 7.3 ± 1.8 4.9 ± 0.3 62.8 ± 7.6 53.3 ± 1.1 
slgM 127.4 ± 11.8 114.2 ± 6.5∗ 205.7 ± 14.0 173.3 ± 24.2 

Isolated splenocytes were treated with LPS or just PBS for 72 h in tissue culture. Cells were counted, and flow cytometry was carried out on the splenocytes as described in Materials and Methods. All rows show mean fluorescence units on B220+ B cells from each marker and each group of mice. Three mice were used per group, and Student’s t test was used to establish p values. ∗, p < 0.05.

We have reported previously the generation of three distinct lines of CR2/CD21-tg mice using a murine Vλ2 promoter/Vλ2-4 enhancer minigene (24). In that study, the hCR2high line was found to exhibit a marked reduction in peripheral B cell numbers, as well as reduction in basal serum IgG levels, and a clear deficit in the T-dependent immune responses to NP-KLH. It was also established in these studies that the presence of endogenous mCR2/CR1 had little effect on the overall B cell numbers or B cell subsets of the hCR2-expressing mice, suggesting that hCR2 expression on the mouse B cell was not superseding or masking any subsequent role for mCR2/CR1. In the studies reported herein, we conducted immune response experiments in the complete absence of mCR1/CR2 to focus on the independent functional effects of premature expression, complement binding, and signaling mediated through hCR2 alone.

We originally predicted that hCR2 expression alone would provide an enhanced or reconstituted humoral immune response when compared with hCR2 mice in the absence of endogenous mCR1/CR2. However, the data presented here show that this was not the case and in fact the opposite was seen, most significantly in the hCR2high mice. These mice displayed a marked reduction in IgG subclass titers (Figs. 1, c–f, and 2, c–f) and GC responses (Fig. 3, a–e), indicative of a substantially muted humoral immune response in response to challenge with SRBC as a model Ag. These data are supportive of our previously published data on mice expressing hCR2 in the presence of mCR1/CR2. In that limited study, we noted that the level of total IgG was reduced significantly in mice expressing the highest levels of hCR2 on their B cells both in unimmunized animals and in response to the T-dependent Ag NP-KLH (24). In the present study, we found that even in the hCR2low mice, which have no obvious B cell numeric defects, the humoral immune response was diminished in several respects.

The key difference between endogenous mCR2/CR1 expression and the hCR2 transgene expression is the stage in B cell development where these molecules are expressed. mCR2/CR1 is first expressed during the IgMhighIgDlow immature cell stage of development (18, 19), whereas these tg mice initiate expression of hCR2 during the CD43+/CD25 late pro-B cell stage of development (24). Thus, hCR2 can potentially interact with ligands during the subsequent critical stages of B cell development in which the B cell is taught the difference between self and nonself. The marked decreases in hCR2 expression levels noted on mature B cells isolated from the bone marrow of hCR2high and hCR2int mice suggests that B cells in these mice control the level of signal received through CR2 by modulating its expression during these stages of B cell development (Table I). Furthermore, the increased levels of the B cell activation markers B7.1 and B7.2 and the even more impressive increases in CD44 and MHC class II (Table 2) indicate that these B cells have received a potent signal during their development. Preliminary Ca2+ flux data suggests that cross-linking of hCR2 with mC3d tetramers provides a robust Ca2+ flux in peripheral B cells isolated from all lines of hCR2-tg mice (data not shown), confirming that hCR2 has the potential to signal the B cell during these critical stages of B cell development. The activation status of B cells in the hCR2high mice appears to be similar to that noted in the modified anti-Smith Ag model of anergy; here, too, MHC class II and anti-Fas were up-regulated on B cells in the periphery (36). These authors concluded that this indicated that these cells had been marked for deletion unless a sufficient survival signal was produced; this may also be the case in the hCR2high mice. Notably, in contrast to the low-affinity anti-Smith Ag anergy model, we find that B cells from the hCR2high line have up-regulated CD44 and B7.1 and B7.2, most notably in the blood. These molecules have been found to be increased in other models of anergy such as the HEL double tg model and the 2-12H-tg mice. Thus, in the hCR2-tg mice, it appears that the B cells have received an activation signal yet have become unresponsive as suggested by the muted humoral immune response and inefficient GC reaction (Figs. 1–3). Therefore, a form of anergy may exist in these mice. Of note, in mice that express hCR2 only on mature B cells, no reduction in B cell numbers is noted and response to SRBCs is enhanced over hCR2 littermates (41).

Anergy is thought to arise as the immature B cells leave the bone marrow and enter the periphery (12, 42, 43). If they meet Ags without the appropriate T cell help (CD40 Ligand), they are anergized or deleted. Evidence from elegant studies where Cr2−/− mice where bred onto the HEL-tg/sHEL mice or C57BL/6lpr/lpr background mice suggests that mCR1/CR2 are critical for establishing and maintaining tolerance, i.e., a signal through mCR1/CR2 directly contributes to deletion or anergy of autoreactive B cells (44). This is further supported from findings in mice that naturally express a mutated form of CR2 that has a reduced ability to bind C3d. This results in a loss of coreceptor activity and a marked increase in the incidence of autoimmune disease (in the form of a lupus-like syndrome) (45). Taken together, these findings suggest that expression of a functioning CR2 molecule alongside BCR on the B cell surface is essential for maintaining peripheral B cell tolerance. Our data is wholly consistent with this suggestion. In the hCR2-tg mice, all the observed defects can be explained by considering that the early expression of CR2 in B cell development heightens the normal tolerogenic role of CR2 in the mouse and thus results firstly in a deletion of cells, which would have normally survived, and secondly induces an anergic-like phenotype on the majority of the remaining B cell population (as outlined in Fig. 4), which interacts only transiently with Ags through the BCR/CR2 complex (or possibly a pre-BCR/CR2 complex).

FIGURE 4.

Model of effect of early expression of hCR2. The developing B cell meets a strong signal through the BCR (or pre-BCR) and undergoes apoptosis (a). The developing B cell obtains an intermediate signal from a soluble Ag and becomes anergic (b). The developing B cell meets a soluble Ag with intermediate binding through the BCR (or pre-BCR) but also gets a signal from CR2, convincing the B cell that it must undergo apoptosis (c). The B cell receives a weak or barely present signal through the BCR (or pre-BCR), but a strong signal from CR2 is enough to alter its activation potential and enter into an anergic-like state (d).

FIGURE 4.

Model of effect of early expression of hCR2. The developing B cell meets a strong signal through the BCR (or pre-BCR) and undergoes apoptosis (a). The developing B cell obtains an intermediate signal from a soluble Ag and becomes anergic (b). The developing B cell meets a soluble Ag with intermediate binding through the BCR (or pre-BCR) but also gets a signal from CR2, convincing the B cell that it must undergo apoptosis (c). The B cell receives a weak or barely present signal through the BCR (or pre-BCR), but a strong signal from CR2 is enough to alter its activation potential and enter into an anergic-like state (d).

Close modal

In the previous study, we found peripheral blood B cell numbers reduced by 60% in the hCR2high mice and by 15% in hCR2int mice, whereas hCR2low mice showed no difference in B cell numbers when compared with hCR2 littermates (24). Notably, splenic B cell numbers were not affected to the same degree, suggesting that maturation of B cells through the spleen also contributes to the reduction in B cell numbers. Indeed, the fact that hCR2 expression levels on B cells isolated from hCR2high and hCR2int mice are further suppressed in the splenic compartment when compared with B cells in the bone marrow suggests that a second round of hCR2 modulation is required to allow these B cells to reach full maturity (Table I). In the hCR2high mouse, the significant increase in B cell expression of CD95 indicates that the signal during B cell development that is derived through hCR2 is sufficient to earmark these cells for deletion by apoptosis. Rescue of B cells from apoptosis using complement-coated Ags has been demonstrated in the WEHI mouse B cell line (23), and thus, if a sufficient signal is received through the BCR, either with or without ligation of hCR2, then these CD95/hCR2-expressing B cells may be rescued from apoptosis. This would partly explain why a high dose of Ag is sufficient to generate an immune response in the hCR2high mice and a slightly increased boost response, even though the humoral immune response is reduced overall. Whether the use of adjuvant or even higher doses of Ags would completely overcome the observed defects remains to be formally tested. However, data from Ags plus adjuvant immunization of the CR2−/− mice suggest that a recovery in immune titers to essentially wild-type levels is possible (30) and certainly merits a detailed examination in the hCR2-tg mice. Despite the reduction in B cell numbers and the obvious functional deficits in the remaining B cell population, the hCR2-tg mice appear healthy, indicating that the deficit does not seriously affect their ability to deal with typical animal house pathogens. Whether they will display reduced ability to counter specific infections remains to be formally tested and will be the focus of subsequent experiments in these mice.

In summary, we show that hCR2-expressing murine B cells do not respond efficiently to the T-dependent Ag, SRBC, both in terms of immune titer and in the generation of an adequate GC response. The level of hCR2 expression is directly proportional to the severity of the immune deficit. B cells isolated from hCR2-tg mice have a marked increase in multiple B cell activation markers and a significant increase in CD95 expression, indicative of an increased potential to undergo deletion through apoptosis. The data suggest that B cells in the hCR2-tg mice have an altered activation state similar in many respects to Ag-specific anergy. Thus, the expression of hCR2 during early B cell development leads not only to a reduction in B cell numbers but also dramatically alters the ability of the remaining B cells to respond to Ags. The mechanism by which expression of CR2 at the pre-B cell stage can result in such far-reaching effects on B cell survival and function remains unclear. Nevertheless, the hCR2-tg mice will provide a useful model to dissect the mechanisms that govern the generation of B cell tolerance both in the bone marrow and the periphery.

We thank Drs. Brad Spiller and Awen Gallimore for their many helpful discussions, Beverley Giddings for guidance with the immunohistochemistry, and the technical staff within the department for helping to facilitate this work.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

These studies were supported by R0-1 AI31105 (to V.M.H.) and a Wellcome Trust Career Development Award (to K.J.M.).

3

Abbreviations used in this paper: tg, transgenic; HEL, hen egg lysozyme; sHEL, soluble HEL; CR2, complement receptor type 2; mCR2, mouse CR2; hCR2, human CR2; GC, germinal center; SA, streptavidin; NP-KLH, 4-hydroxy-3-nitrophenyl-keyhole limpet hemocyanin.

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