Expression of Human Complement Receptor Type 2 (CD21) in Mice During Early B Cell Development Results in a Reduction in Mature B Cells and Hypogammaglobulinemia

Kevin J. Marchbank, Liudmila Kulik, Matthew G. Gipson, B. Paul Morgan and V. Michael Holers
J Immunol October 1, 2002, 169 (7) 3526-3535; DOI: https://doi.org/10.4049/jimmunol.169.7.3526

Abstract

Complement receptor (CR) type 2 (CR2/CD21) is normally expressed only during the immature and mature stages of B cell development. In association with CD19, CR2 plays an important role in enhancing mature B cell responses to foreign Ag. We used a murine Vλ2 promoter/Vλ2–4 enhancer minigene to develop transgenic mice that initiate expression of human CR2 (hCR2) during the CD43+CD25− late pro-B cell stage of development. We found peripheral blood B cell numbers reduced by 60% in mice expressing high levels of hCR2 and by 15% in mice with intermediate receptor expression. Splenic B cell populations were altered with an expansion of marginal zone cells, and basal serum IgG levels as well as T-dependent immune responses were also significantly decreased in transgenic mice. Mice expressing the highest levels of hCR2 demonstrated in the bone marrow a slight increase in B220intCD43+CD25− B cells in association with a substantial decrease in immature and mature B cells, indicative of a developmental block in the pro-B cell stage. These data demonstrate that stage-specific expression of CR2 is necessary for normal B cell development, as premature receptor expression substantially alters this process. Alterations in B cell development are most likely due to engagement of pre-B cell receptor-mediated or other regulatory pathways by hCR2 in a CD19- and possibly C3 ligand-dependent manner.

B cell development from multipotent stem cells through to Ig-secreting plasma cells is marked by the differential expression of surface molecules, many of which influence cell fate. Regulation of early B2b cell development occurs primarily in the bone marrow and is tightly regulated by interactions with stromal cells, the expression of the pre-B cell receptor (BCR)3 and its associated signaling molecules, B cell:B cell homotypic adhesion, and the cytokine IL-7 (1, 2, 3, 4). The use of gene targeting and transgenic technology has demonstrated the importance of many of the molecules that play an essential part in B cell development and function from B cell progenitors through to mature circulating B cells.

Human complement receptor (hCR) type 2 (hCR2/CD21) is one such B cell molecule whose expression is regulated during development. Complement receptor (CR)2 is a receptor for the C3d activation fragment of the complement component C3 (5) in addition to the larger C3dg and iC3b fragments (6, 7). CR2 is also a receptor for the EBV through interactions with the surface glycoprotein gp350/220 (8, 9). In addition, CR2 serves as a receptor for CD23 (10). Expression of hCR2 on B lymphocytes was first studied by Tedder et al. (11), who showed that the receptor was not found on pre-B or late stage plasma cells, but was readily detectable on mature B cells. Mouse (m)CR2 was also found to display this developmentally regulated expression pattern with receptor expression first found on immature B cells, and increasing as cells mature into transitional, follicular, and marginal zone cells (12). Of interest, the onset of mCR2 expression during development has also been shown to be highly correlated with the transition from editing to deletion in the response of newly developing immature B cells to self Ag (13).

The human and mouse receptors are very similar, as hCR2 and mCR2 demonstrate 67% identity at the nucleotide level and 58% identity in the protein sequence (14, 15). Both receptors exhibit a Mr of ∼140–150 kDa, and hCR2 binds mouse and hC3d with indistinguishable affinities (16). In mice, both mCR1, which has an additional binding site for C3b and C4b, and mCR2 receptors are derived by alternative splicing from a common mRNA (14, 15). The protein structure of CR2 is dominated by a phylogenetically ancient structural motif designated the short consensus repeat (SCR). The extracellular domain of both hCR2 and mCR2 is comprised entirely of 15–16 tandemly arranged SCRs, followed by a transmembrane domain and a short intracellular domain. The C3d ligand binding domain of both receptors is found within the N-terminal two SCRs (16, 17, 18, 19).

CR2 is one of several important coactivators of B cells. The ability of CR2 to enhance mature B cell activation in response to the binding of Ag containing covalently attached C3d was first suggested in studies using mAbs (20) as well as Ag coupled to multiple copies of C3d (21) to coligate CR2 with the BCR. The importance of these results was confirmed in two independent studies in which mice were generated by insertional mutagenesis of the Cr2 gene, resulting in a deficiency of mCR2/CR1 (22, 23, 24). Consistent with the postulated role for CR2, Cr2−/− mice demonstrate a marked decrease in humoral immune responses to T-dependent Ags as well as impaired germinal center formation (25, 26) and B cell memory (27, 28). CR2/CR1-deficient mice exhibit no known abnormalities in developing B cell populations in the bone marrow during development, and splenic, lymph node, and peripheral blood B2b cell numbers are unaltered. B1b cells were reported to be decreased in one strain of Cr2−/− mice (22), although the mice used in the studies in this work have consistently not demonstrated this phenotype (24) (data not shown).

Although CR2 has a short cytoplasmic tail that does not have known signaling capabilities, it nevertheless exerts potent B cell signaling effects through its noncovalent association with CD19 and CD81 (TAPA-1) (29, 30). Gene-targeting experiments that have eliminated expression of CD19 or CD81 illustrate the importance of these molecules and the CR2/CD19/CD81 complex in humoral immunity (31, 32, 33, 34). CD19 is expressed relatively early in B cell development, at the time of μ H chain gene rearrangement (35, 36). Cd19−/− mice exhibit no discernible change in early B cell development in the bone marrow, and similar to Cr2−/− mice, the major functional defects in Cd19−/− mice are found in immature and mature B cells in response to foreign and self Ag. In addition to a humoral immune defect, Cd19−/− mice demonstrate a marked reduction in B cell numbers in the spleen and peripheral lymphoid tissue as well as a marked decrease in B1b cells in the peritoneum (31, 33). Thus, the phenotype observed in Cd19−/− mice is considered to be similar to, but more severe than that observed for Cr2−/− mice.

hCD19 transgenic mice (37), which express hCD19 at the appropriate stage of mouse B cell development, but at much higher levels than endogenous mCD19 expression, display major defects in B cell development. B cell numbers are markedly reduced from the immature B cell stage forward. In contrast, although, the numbers of B1b cells are increased in both the peritoneum and spleen, consistent with a role in positive selection of those cells. Importantly, hCD19 expression also results in the development of serum autoantibodies and loss of tolerance to self Ags (36, 38).

To further investigate the role of CR2 in B cell development, as well as create a model of hCR2 expression in primary cells in which structure-function relationships could be studied in vivo, we have created mice expressing hCR2 cDNA under the control of a B cell-specific promoter/enhancer minigene that initiates transgene expression in the late pro-B cell stage (also designated pre-BI or stage B/C) (1, 39, 40, 41). We found expression of hCR2 on mouse B lymphocytes by flow cytometric and RT-PCR analysis in several founders, and then established three lines of mice classified as expressing high, intermediate, or low levels of the human receptor. Expression of high levels of hCR2 from the late pro-B cell stage on resulted in a 60% reduction in B cell numbers in the periphery and substantial alterations in bone marrow B cell development indicative of a developmental block at the pro-B cell stage. In addition, splenic B cell populations were altered, and basal serum IgG levels as well as T-dependent humoral immune responses were decreased. These data strongly suggest that the presence of high levels of hCR2 on mouse B cells at inappropriately premature stages of maturation results in a significant alteration in B cell development and peripheral B cell immune responses, most likely by engagement of regulatory pathways important in maintaining coordinated B cell development.

Materials and Methods

Cells

Blood was collected into 20 μl heparin following a tail vein nick and washed once in cold PBS. Human PBL were isolated from 40 ml blood from a healthy donor by density centrifugation on Histopaque (Sigma-Aldrich, St. Louis, MO). Bone marrow B cells were collected by flushing mouse femurs with cold PBS. Peritoneal B cells were isolated by injection of 5–10 ml PBS into the peritoneal cavity. PBS was then extracted after 1–2 min, and macrophages were adsorbed out by incubation on plastic plates three times at room temperature (RT) for 5 min before staining. Isolated spleens were disrupted by pressing between frosted glass slides in PBS buffer and transferred to 15-ml 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, 0.02% sodium azide). All murine samples were incubated with 0.5–1 ml RBC lysis buffer (0.83% NH4Cl, 0.1% KCO3, 0.1 mM EDTA) and incubated at RT for 1–2 min. The cells were then washed with 1 ml staining buffer. Cells were then counted, and 1–3 × 106 cells/ml were used per analysis. Cells were stained as described below.

Antibodies

Purified and biotin-conjugated mAb 171 (anti-hCR2, IgG1 isotype) (42), IgG1 isotype control, and the polyclonal anti-hCR2 Ab were produced in the laboratory following standard methods. The THB-5 anti-CD21-producing hybridoma line was obtained from the American Type Culture Collection (Manassas, VA), and the HB-5 anti-hCR2 mAb was purified and biotin conjugated using standard techniques. The 2.4G2 (anti-mCD16/mCD32, FcBlock), PE-conjugated B-Ly-4 (anti-hCR2), biotin- or FITC-conjugated 145-2C11 (anti-CD3ε), FITC-conjugated RA3-6B2 (anti-mCD45R, B220), PE-conjugated 6C3 (anti-6C3/BP-1 Ag), PE-conjugated S7 (anti-CD43, Ly-48, leukosialin), biotin-conjugated anti-CD24 (heat-stable Ag (HSA)), PE-conjugated anti-CD5 (Ly-1), biotin-conjugated anti-CD25 (IL-2Rα chain), biotin-conjugated anti-CD23, PerCP-conjugated anti-B220, FITC-conjugated anti-CD24, and streptavidin (SA)-allophycocyanin were all obtained from BD PharMingen (San Diego, CA). FITC-conjugated HD37 (anti-hCD19) was obtained from DAKO (Carpinteria, CA). SA-FITC-, SA-PE-, and rhodamine-conjugated donkey anti-rabbit Ig were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). FITC-conjugated anti-IgM, biotin-conjugated anti-CD11b (Mac-1), purified goat anti-mouse IgG/IgM, alkaline phosphatase (AP)-conjugated goat anti-mouse IgG, and goat anti-mouse IgM were obtained from Caltag Laboratories (Burlingame, CA).

Creation and screening of transgenic mice

To create transgenic mice, the hCR2 cDNA was released from the pSFFV-neo vector (17, 43) using EcoRI and inserted into the Vλ2 promoter/Vλ2–4 enhancer minigene (44, 45), a generous gift from J. Hagman (National Jewish Medical and Research Center, Denver, CO). Orientation was confirmed by PCR and restriction endonuclease analysis. The hCR2-encoding minigene was released from the pUC19 plasmid backbone using SalI and separated from the vector using low melt point agar (FMC Bioproducts, Rockland, ME). The purified DNA was then injected into fertilized mouse OVA isolated from FVB/N mice by the University of Colorado Health Sciences Center Transgenic Core Facility using standard techniques.

To screen for the hCR2 transgene in potential founder 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% Nonidet P-40 v/v, 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 detergent lysis supernatant. Confirmed founders were subsequently backcrossed onto the Cr2−/− or Cr2+/+ C57BL/6 strain and followed by PCR using two independent primer sets (set 1, Vλ2-hCR2, 5′-gtgaattaaggcctggacttcactt-3′ and 5′-TTATCCCAGGTTCCATCCAC-3′, a set that goes across the Vλ2-hCR2 cloning junction; and set 2, 5′-ATGAGGGGCAGGTTGGCTCC-3′ and 5′-GGCAGGAAAACTTTCTATATGG-3′), primers within the hCR2 cDNA sequence at cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. Use of both primer sets confirms that the transgene vector and the insert are both present and in the correct orientation. Both PCR were conducted for 27 cycles. Genotyping was also confirmed by flow cytometric analysis. F1 to F5 backcross mice as designated were used in these studies.

Estimation of transgene copy number

Estimation of relative hCR2 transgene copy number in transgenic mice was conducted by PCR analysis. A known quantity of hCR2 transgene plasmid DNA in nontransgenic murine DNA was used to establish a standard curve for copy number. Bone marrow cells were isolated as described above. A known quantity of bone marrow cells was digested in detergent lysis buffer, as described above. Three microliters of supernatant were used in each PCR, as described above for genotyping. Products were separated on agarose gel, and densitometry was used to calculate the copy number relative to the standard curve. The first primer sets outlined above were used, and several members of each transgenic line were examined to control for PCR and sample variation.

Immunofluorescence

Mouse tissue was collected, mounted in OCT compound (EMS Laboratories, Ashford, U.K.), and snap frozen in isopentane. Frozen sections were cut on a cryotome (Shandon; Life Sciences International Europe, Runcorn, U.K.). Where appropriate, biotin and avidin binding sites were blocked using a biotin/avidin blocking kit (Vector Laboratories, Peterborough, U.K.). Sections were incubated with 1/100 B220 FITC, rabbit anti-hCR2 IgG, anti-CR2 mAb 171, or biotin-conjugated anti-mCD3 in 1% BSA/PBS for 1 h at RT. Slides were washed three times with PBS. Where appropriate, slides were incubated with 1/200 FITC-conjugated goat anti-mouse IgG1 Ab (Southern Biotechnology Associates, Birmingham, AL), SA-FITC, or rhodamine-conjugated donkey anti-rabbit Ig in addition to 4′,6-diamidino-2-phenylindole (nuclear stain, 0.1 pg/ml in PBS) for 1 h at RT. Slides were washed three times in PBS. Surplus buffer was removed, and a drop of Vectashield (Vector Laboratories) was added to sections. Sections were viewed using a Leica (Deerfield, IL) microscope with attached digital camera, and analyzed using Improvision Openlab software (Improvision, Coventry, U.K.).

Flow cytometry

After RBC lysis, cells were washed and then resuspended in 10 μg/ml 2.4G2 mAb to block FcR. After a 15-min incubation on ice, cells were washed in staining buffer (1% BSA/0.01% NaN3/PBS) and resuspended in 100 μl staining buffer containing primary Ab (0.1–3 μg/ml) and 1 μl anti-B220 FITC or 5 μl anti-IgM FITC, where appropriate. Cells were incubated for 30 min on ice in the dark. After incubation, cells were washed in staining buffer three times and then incubated with the appropriate SA-conjugated fluorochrome to detect biotin-labeled primary Abs. Following incubation, cells were washed as above and then resuspended in staining buffer containing 1% formaldehyde. Flow cytometry was carried using a BD Biosciences FACSCalibur (Oxford, U.K.).

Ig ELISA

Serum was collected from 8- to 12-wk-old F4 mice by tail bleed. ELISA plates were coated overnight with goat anti-mouse IgG/IgM (H + L) in a carbonate buffer and blocked with 200 μl 1% BSA/PBS/0.02% Tween 20 for 1 h. Mouse Ig reference serum (Bethyl Labs, Montgomery, TX) or transgenic mouse serum was diluted in blocking buffer and incubated on the plate for 2 h at 37°C. Plates were washed three times with PBS, followed by a 1-h incubation with blocking buffer. The appropriate AP-conjugated Ab (1/3000) was then incubated on the plates for 1 h at 37°C. Plates were washed with PBS, and 100 μl substrate solution (ImmunoPure p-nitrophenyl phosphate disodium salt; Pierce, Tattenhall, U.K.) tablets were dissolved in diethanolamine substrate buffer to create a 1 mg/ml solution of p-nitrophenyl phosphate disodium salt. Concentration of Ig isotype was calculated using the standard curve generated from the reference serum. No fewer than six animals per group were used, and each serum was measured in quadruplicate.

Immunization and Ag-specific ELISA determination

Mice were immunized by the i.p. route with 200 μl PBS containing 50 μg nitrophenyl (NP)36-keyhole limpet hemocyanin (KLH; Biosearch Technologies, Novato, CA). Serum was obtained before and at the indicated intervals following immunization. Serum anti-NP levels were measured by coating Immulon 1B plates (Dynatech, Chantilly, VA) with 5 μg/ml NP17-BSA (Biosearch Technologies) in PBS. Bound Ab was detected using 0.2 μg/ml AP-conjugated anti-mouse IgG Ab (Caltag Laboratories), followed by p-nitrophenyl phosphate (Sigma-Aldrich) at 1 mg/ml. The mean OD405 for samples was compared with a standard curve of titrated hyperimmune serum to calculate relative units.

Results

Creation of transgenic mice expressing hCR2

A well-characterized cDNA encoding the full-length 15 SCR form of hCR2 (17, 43) was cloned into a previously used mouse Vλ2 promoter-Vλ2–4 enhancer minigene (44, 45) (Fig. 1⇓), and the Vλ2 promoter/hCR2 cDNA/Vλ2–4 enhancer containing insert of the plasmid was injected into fertilized mouse OVA using standard techniques. This promoter is active in the late pro-B cell phase before the onset of endogenous L chain transcription (44). PCR analysis of ear punch DNA from the resulting pups revealed the presence of seven genotype-positive mice. Analysis of PBL was conducted by flow cytometry and demonstrated that five of the seven mice expressed hCR2, with each expressing the transgenic receptor only on B cells. In these founders, subsequent analysis at sacrifice of spleen, mesenteric lymph node, bone marrow, and peritoneal cavity-derived cells confirmed that only B220+ cells expressed hCR2 (data not shown).

FIGURE 1.
FIGURE 1.

Diagram of the Vλ2 promoter/hCR2 cDNA/Vλ2–4 enhancer minigene. Shown is the λ L chain minigene and the position of the hCR2 cDNA relative to the promoter and enhancer. Also shown are the SalI restriction endonuclease sites for removal of the minigene from the pUC19 plasmid backbone and the position of the PCR primers (sets 1 and 2; see Materials and Methods) used to detect the transgene.

All B cells isolated from the five original founders expressed hCR2, indicating that the transgene was active regardless of endogenous H or L chain usage. These transgenic mice were then divided into three categories according to hCR2 expression: those with a high (hCR2high; one founder), intermediate (hCR2int; two founders), or low (hCR2low; two founders) level of hCR2 expression on their B cells (Fig. 2⇓, A–D). One founder was chosen from each group and bred with Cr2+/+ or Cr2−/− mice that were on the C57BL/6 background at the F7 backcross (24, 46) to create mice expressing hCR2 in either the presence or absence of mCR2/CR1. Notably, as found in our previous studies (46), mCR2/CR1 expression levels were unaltered regardless of the level of hCR2 expression on mouse B cells at the F1 backcross to Cr2−/− C57BL/6 mice (Fig. 2⇓, A–D) or on the Cr2+/+ C57BL/6 background (data not shown). Levels of hCR2 expression on mouse B cells in the spleen and blood have remained consistent and comparable with each other through subsequent F1-F5 backcrosses to either Cr2−/− or Cr2+/+ mice.

FIGURE 2.
FIGURE 2.

Expression of hCR2 on peripheral blood B cells isolated from transgenic mice. A–D, PBL were isolated from F1 mice on the Cr2−/− backcross and then stained with B220 FITC to identify B cells and biotinylated HB5 (anti-hCR2; filled), 7E9 (anti-mCR2; black line), or mIgG1 isotype control (dashed line), followed by SA-PE to evaluate CR2 expression levels. Shown are B220+ cells only. E, Normal human PBL and mouse splenocytes were analyzed by flow cytometry. Cells were identified as B cells both through their forward and side scatter profile as well as B220+ phenotype (mouse B cells) or CD19+ phenotype (human B cells). Cells were stained with anti-hCR2 (B-Ly-4-PE) or isotype control from the same diluted stock and read on the same day using the same cytometer settings. Isotype control on human B cells (gray dashed line); B-Ly-4-PE staining of splenocytes isolated from hCR2− (thin black line), hCR2int (thick gray line), hCR2high (thick black line), and human B cells (gray filled). At least 5000 events were collected, and results are representative of triplicate analyses.

Expression of hCR2 is appropriately tissue restricted

Direct comparison of mouse B cells with peripheral human B cells using flow cytometry indicated that hCR2 expression levels on hCR2high mice were ∼25% of that seen on normal human B cells (Fig. 2⇑E). Therefore, while there is substantial expression of hCR2 in transgenic mice, it is not greatly increased relative to endogenous mCR2/CR1 receptor levels. Likewise, hCR2 expression levels on B cells isolated from hCR2int mice were found to be ∼15% of that on normal human B cells, and on hCR2low B cells proportionately less (data not shown).

Estimation of transgene copy number indicated that the relative level of hCR2 protein expression correlated with the relative copy number (Fig. 3⇓). This result strongly suggests that increasing transgene copy number is responsible for different hCR2 expression levels noted in the three lines. Analysis of progeny in each line revealed a Mendelian inheritance pattern, consistent with a single insertion site (data not shown). Immunofluorescence analysis of thymic and nonlymphoid tissues was used to further confirm that hCR2 expression was restricted to B cells (Fig. 4⇓). B cell follicles with cells expressing hCR2 are clearly visible in the spleen section from hCR2high mice (Fig. 4⇓, A, C, and E), as compared with splenic sections from nontransgenic (hCR2−) littermates (Fig. 4⇓, B, D, and F). As expected, no detectable hCR2 protein expression was found in any of the other tissues examined in transgenic mice using the highly specific anti-hCR2 mAb 171 (Fig. 4⇓, G–L).

FIGURE 3.
FIGURE 3.

Transgene copy number is proportional to expression of hCR2 on mouse B cells. Bone marrow cells were collected and lysed to release genomic DNA. PCR was conducted on known copy number quantities of plasmid DNA containing the hCR2 transgene and compared with the intensity of the PCR product generated from genomic DNA isolated from known numbers of bone marrow cells. Results are representative of three independent experiments.

FIGURE 4.
FIGURE 4.

Immunofluorescence analysis demonstrates that hCR2 has a restricted tissue distribution and is expressed on B cells. Tissues were collected from hCR2high and hCR2− mice. Splenic sections A (hCR2high) and B (hCR2−) were stained with B220 FITC to label B cells and a polyclonal anti-hCR2 Ab, followed by rhodamine-conjugated secondary Ab. Colocalization (yellow stain) of both Abs is seen only in section A. Splenic sections C (hCR2high) and D (hCR2−) were stained with biotin-conjugated anti-CD3, followed by SA-FITC to label T cells in addition to a polyclonal anti-hCR2 Ab, followed by rhodamine-conjugated secondary Ab. These results demonstrate unique staining of B and T cells. Splenic sections E (hCR2high) and F (hCR2−), as well as sections G–L (all hCR2high) were stained with anti-hCR2 mAb 171, followed by goat anti-mouse IgG1 FITC. All sections were stained with 4′,6-diamidino-2-phenylindole (nuclear stain). Shown are tissue sections from thymus (G), kidney (H), liver (I), heart (J), lung (K), and brain (L).

Peripheral B cell numbers are significantly reduced in hCR2high and hCR2int mice

During initial phenotyping experiments, it became evident that B cell numbers were substantially altered in hCR2 transgenic mice. Analysis of PBL isolated from hCR2high mice showed a 60% reduction in B cell numbers (B220+) when compared with hCR2− littermates (Table I⇓). The average age of mice examined was between 8 and 12 wk, and the results are inclusive of F1-F5 mice on either a Cr2+/− or Cr2−/− C57BL/6 background, as shown. PBL isolated from hCR2int mice demonstrated a 15% reduction in B cell numbers when compared with hCR2− littermates. PBL isolated from hCR2low mice displayed no change in the B cell numbers. These data indicate that hCR2 expression levels are inversely related to the observed reduction in B cell numbers. Importantly, the presence or absence of mCR2 did not influence the effect of hCR2 expression level on the reduction of B cell numbers.

View this table:
Table I.

Transgenic mice demonstrate a marked reduction in peripheral B cell numbers and altered splenic composition

Analysis of splenocytes isolated from the hCR2high mice displayed a 20% reduction in the B cell population (Table I⇑). This was associated with a 15% reduction in spleen weight in hCR2high mice compared with hCR2− littermates (data not shown). No significant alteration in splenocyte number or spleen weight was observed in the hCR2int mice. Examination of CD19 and surface IgM expression levels on splenocytes isolated from hCR2high B cells showed no quantitative differences when compared with hCR2− littermates (data not shown). Examination of the relative percentage of λ and κ L chain-bearing B cells in the spleen revealed no difference (data not shown). Analysis of the peritoneal B cell populations of hCR2high mice indicated that the B2b cell (or conventional B cell) population was unaltered in this compartment (Table I⇑), and total peritoneal cell numbers were unchanged overall. However, a trend to a small increase in B1a and decrease in B1b cell numbers in hCR2high and hCR2int mice compared with hCR2− littermates (Table I⇑) was observed; however, this was not statistically significant. Notably, the presence or absence of mCR2 did not alter the findings with regard to B1a and B1b cell numbers (data not shown).

Immature and mature B cell numbers are substantially decreased in hCR2 transgenic mice

Flow cytometric analysis of hCR2high bone marrow-derived cells also revealed a marked decrease in the overall percentage of B cells as compared with hCR2− littermates. Developing B cells were then examined using stage-specific markers according to the method of Hardy et al. (1, 39). First, consistent with the reported transcriptional activity of this λ minigene (44), we found that hCR2 itself was detectable on the B220int CD43+CD25− population of B cells in hCR2high mice (Fig. 5⇓A). Note that the difference in B220-staining pattern in this figure from that normally seen reflects alterations in B cell development described below. Transgene expression from this point of development on was also found in the hCR2int and hCR2low trangenic mice (data not shown). Interestingly, expression levels of hCR2 on these B220int early progenitor B cells appeared on average to be higher than that seen on mature B cells (Fig. 5⇓B).

FIGURE 5.
FIGURE 5.

B cells expressing high levels of hCR2 are blocked in the late pro-B stage of B cell development. Bone marrow cells were isolated by flushing the femurs of mice with cold PBS. A, Flow cytometry was conducted on bone marrow cells isolated from hCR2high mouse that had been stained for B220 and CD25, CD43, or hCR2. Forward and side scatter profile was used to identify the large proliferating B lymphocyte population (R1) and the small resting B lymphocyte population (R2) for subsequent analyses. Results demonstrate expression of hCR2 on the B220intCD43+CD25− population. B, Bone marrow cells isolated from hCR2 transgenic mice as noted in addition to P1-5, a previously described hCR2 transgenic mouse created using the entire human gene (47 ), and hCR2− control mice were stained for B220 and hCR2. Cells falling into combined R1 and R2 gates are shown. C, Bone marrow cells were isolated from hCR2high, hCR2int, and hCR2− Cr2+/+ mice. Isolated cells were stained for B220 and mCR2, CD43, CD25, or BP-1. Cells falling into combined R1 and R2 gates are shown. In all cases, 100,000 B220+ events were collected and density plot analysis was performed. Histograms shown are representative of data collected from at least four mice in each group. Quantitative comparisons of flow cytometric analyses that include all bone marrow cells are found in Table II⇓.

As an apparent consequence of expression of hCR2 at this point of development, relative B220intCD43+CD25− B cell numbers were slightly increased in hCR2high mice in comparison with hCR2− littermates (Table II⇓). Conversely, later developing B cell populations, B220intCD43− pre-B and immature B cells as well as B220highCD43− mature B cells, showed a substantial decrease in hCR2high mice when compared with hCR2− littermates, with a mean reduction of ∼50% in each group. Analysis of the hCR2int mice revealed no overall increase in B220intCD43+CD25− B cells; however, a modest decrease in B220intCD43−CD25+ cells was noted. These results are indicative of a developmental block at the pro-B cell stage, most apparent in the hCR2high mice, but also present in hCR2int transgenic mice. Representative examples of these changes are shown in Fig. 5⇑C, in which further analysis of BP-1 expression also confirms the block in development at this stage.

View this table:
Table II.

hCR2high transgenic mice display an increase in pro/pre-B cells and a marked decrease in later stage B cellsa

The assignment of B cells to the appropriate B cell stage was confirmed by use of Abs to IgM and IgD (data not shown). The presence or absence of mCR2/CR1 did not alter this phenotype (data not shown), and expression of hCR2 did not alter the expression pattern or relative expression levels of mCR2/CR1 (Fig. 5⇑C). Analysis of hCR2low bone marrow showed no significant changes in B cell maturation (data not shown), and expression of hCR2 in the hCR2low mice was found to be comparable with our previously generated hCR2 transgenic mice created using the entire human gene (46) (P1-5; Fig. 5⇑B). In marked contrast to the λ promoter-enhancer mice described in this work, expression of hCR2 in the P1-5 mice was found to be at the appropriate developmental stage for endogenous mCR2 and hCR2, that specifically being during the transition from pre-B cells to immature B cells (Fig. 5⇑B).

Baseline serum IgG levels are decreased in hCR2high mice

The reduction in B cells in the periphery and the alteration in the B cell development pathway seen in the hCR2high mice suggested that immune function would be modified in these mice. Analysis of baseline levels of IgG by ELISA demonstrated a ∼60% reduction in levels (Table III⇓) in the hCR2high mice as compared with hCR2− littermates. Total IgG levels were not significantly altered in the hCR2int mice compared with Cr2−/− mice. The alterations noted above indicate that B cells from the hCR2high mice may fail to progress to normal production of IgG Abs. Of interest, IgM levels were equivalent in hCR2high mice as compared with hCR2− littermates (data not shown), consistent with the relatively normal numbers of B-1 cells as shown in Table I⇑.

View this table:
Table III.

hCR2high mice display a significant decrease in baseline IgG levelsa

T-dependent humoral immune response is decreased in hCR2high transgenic mice

We reasoned that if the primary effect of premature expression of CR2 during B cell development led to hyporesponsive cells, analysis of the T-dependent humoral immune response would demonstrate a decrease. Conversely, if overexpression of hCR2 in peripheral B cells resulted in enhanced coreceptor function similar to that found using the hCR2 gene to drive appropriate stage-specific expression (46), the T-dependent response would be enhanced. To address this question, we immunized hCR2high mice as compared with hCR2− littermates with NP-KLH. Notably, the Ag-specific IgG, but not IgM response was substantially diminished (Fig. 6⇓). These results demonstrate that the B cells that do develop in these transgenic mice have a diminished response to foreign Ag and suggest the presence of a decreased signaling capacity.

FIGURE 6.
FIGURE 6.

T-dependent IgG humoral immune response is decreased in hCR2high mice. Shown by ELISA are IgM (A) and total IgG (B) anti-NP-BSA responses 20 days following immunization with NP-KLH. Nontransgenic (wild-type, n = 13) and hCR2high mice (transgenic, n = 9) were studied. An identical relative decrease in hCR2high mice was noted at days 10 and 30 following immunization (data not shown).

Discussion

We have previously shown that expression of hCR2 at the appropriate stage of B cell development confers partial recovery of the defective immune response in Cr2−/− mice without changing peripheral B cell numbers (46). In that study, the whole hCR2 gene was used to create transgenic mice. Although the human gene is transcriptionally active during appropriate stages of development, confirming previous studies using candidate regulatory regions of the human gene to drive reporter gene expression in transgenic mice (47) and a partial reconstitution of the T-dependent humoral immune response in transgenic Cr2−/− mice was achieved (46), the difficulty of introducing site-specific mutations restricted the usefulness of this particular strategy for structure-function studies. In an effort to produce mice expressing hCR2 at substantially higher levels on their B cells, we used an alternate approach of cloning the hCR2 cDNA into a more commonly used promoter-enhancer minigene (44).

Using this strategy, differences in hCR2 expression levels on mouse B cells were detected by flow cytometry, which allowed these hCR2 transgenic lines to be segregated into three groups demonstrating high, intermediate, and low expression of hCR2. Analysis of copy number of the hCR2 transgene in these mice lines showed the protein expression to be correlated to transgene copy number. Expression of hCR2 was confined to B cells and B cell areas in the spleen, as determined by flow cytometry and immunofluorescence analysis, indicating that, as expected, hCR2 expression is restricted to the B cell lineage by this λ promoter-enhancer minigene. Previous studies using L chain transgenes have also found this to be the case (44). RT-PCR analysis further confirmed that hCR2 is expressed only in B cell-containing tissues (data not shown). Using flow cytometric analysis, the expression of hCR2 protein on mouse splenic B cells from the hCR2high transgenic line was found to be equivalent to ∼25% of the levels detected on normal human PBL, demonstrating that this strategy had not caused an inappropriately large overexpression of hCR2 on mouse peripheral blood or splenic B cells.

We found that B cell numbers were significantly reduced in the bone marrow, peripheral blood, and spleen of hCR2high mice, and decreased in the peripheral blood of hCR2int mice. This was associated in the bone marrow with significantly decreased numbers of B cells in later stages of development in both hCR2high and hCR2int mice. There are several possible explanations that might account for the findings in the bone marrow. Given the requirement for pre-BCR expression, IL-7 cytokine effects, homotypic interactions, and the expression of other signaling proteins in early B cell development, we believe that any explanation should be consistent with known effects of manipulation of these molecules. First, we propose that hCR2 (or presumably mCR2 if it was available) may associate with CD19 during early B cell development and enhance a signal through the pre-BCR (IgH/λ5/VpreB) or its associated signaling complex in a similar manner to that seen with the CR2/CD19 complex and BCR on mature B cells. Second, CR2 could alter homotypic interactions of early developing B cells in a manner previously shown for mature B cells, thus disrupting important cell-cell interactions. And third, CR2 could interact directly with stromal cells and trap the B cell in a particular site that does not allow appropriate B cell development. Any of these possible explanations could be dependent or independent of CR2 interactions with its C3 or CD23 ligands.

With regard to the first possibility, in our previous study we demonstrated that, like mCR2, hCR2 could associate with mCD19 on a mature B cell line and that hCR2 could enhance the T-dependent immune response in Cr2−/− mice (46), which is a CD19-dependent phenotype. In addition, cross-linking of hCR2 and the BCR on splenic B cells isolated from hCR2high mice using biotinylated rC3d (48) and biotin-conjugated anti-BCR mAb b-7-6 produces a pronounced hCR2-dependent increase in intracellular calcium (data not shown). Thus, hCR2 retains the capacity to signal through the CD19/CD81 complex on mature B cells and most likely during earlier stages of development. In addition, with regard to this possibility, previous studies have shown that cross-linking mCD19 with the pre-BCR can enhance intracellular calcium levels (35). CD19 cross-linking has also been shown to block IL-7 down-regulation of RAG-1 and RAG-2 in cultured human progenitor B cells (49), suggesting that hCR2 might also cause similar effects in vivo.

Either of these processes could require hCR2 to bind ligand to exert a biologic effect. Alternatively, hCR2 expression could alter the tonic signal through the early B cell signaling complex in a manner suggested for CD19 and other molecules independently of known ligand binding (1, 2, 3, 50, 51).

Also relevant to the potential role of CD19 interactions in this phenotype is a direct comparison of these results with the h19-1 (hCD19) transgenic mice generated by Zhou et al. (37) and hCR2high transgenic mice described in this work. The high copy number h19-1 transgenic line and hCR2high mice show substantial similarities. Both the hCR2 and hCD19 transgenic mice were bred onto the C57BL/6 strain for analysis, which allows a greater degree of confidence to be drawn from the comparison of phenotype between these transgenic mice. First, there is a remarkably similar level of B cell reduction in the peripheral blood. The expression of hCD19 on mouse B cells also led to a sharp reduction in immature and mature B cells in the bone marrow. Again this effect is found, but to a lesser extent, in hCR2high transgenic mice. Thus, both hCR2 and hCD19 transgenes dramatically alter, but do not completely block B cell development. The major difference between the phenotypes of mice expressing the hCR2 and hCD19 transgenes is that hCR2 transgenic expression significantly modified the late pro-B cell compartment, whereas the hCD19 transgene was not reported to do so. Additionally, hCR2high mice displayed a marked reduction in serum IgG levels, whereas hCD19 transgenic mice demonstrate elevated serum IgG levels.

With regard to the second possibility, that hCR2 alters homotypic adhesion, previous studies have shown that engagement of CR2 promotes homotypic adhesion of B cell lines (29, 52), most likely in a CD19/CD81-dependent manner. Based on these findings, and the postulated importance of B cell:B cell homotypic interactions during bone marrow development (2), it is possible that the binding of ligand to hCR2 could alter this process in a detrimental manner.

With regard to trapping of B cells by interaction with stroma, mAb inhibition studies have previously shown that CR2 is involved in mediating strong adhesion of CR2-expressing B cells to bone marrow stromal cells by interacting with CD23 (53). In the hCR2high mice, therefore, B cells may be inappropriately held to bone marrow stromal cells. In addition, it is intriguing in this regard to note that CD19 itself has been shown to bind stromal cells through interactions with surface-associated heparan sulfate/heparin (54), and this process might also be influenced by hCR2 coexpression.

Less evidence is available to support the possibility that hCR2 expression diminishes the expression or function of other critical B cell molecules. Expression of hCR2 occurs in CD43+ cells before pre-BCR formation, but in a stage when Ig-α and Ig-β are already found on the cell surface (1, 41, 51). Progression from a stage marked as CD43+ to CD43− in mouse B cell development has been affected in a number of studies in which the expression of key molecules has been eliminated by either natural mutations or gene-targeted homologous recombination. These include SCID mice as well as Ig-β, λ5, and RAG-1 or RAG-2 knockout mice (49, 55, 56, 57, 58, 59). Interestingly, λ5−/− mice demonstrate an increase in pro-B cell numbers reminiscent of that seen in the hCR2high mice. A leaky block in B cell development has also been observed in CD24/HSA-deficient mice (60), indicating that removal of this potential signaling element results in B cell maturation defects similar to that seen in the hCR2high mice. Thus, although direct links between these molecules and CR2 have not been previously shown, similarities in phenotypes in mice suggest a possible functional relationship.

In contrast to B-2 development, peritoneal B1a and B1b B cell populations remain relatively unaffected regardless of the level of hCR2 expression. The trend to an increase in B1a B cell numbers that was detected in the hCR2high mice suggests a possible positive effect on the selection of B1a cells similar to that seen with hCD19 trangenic mice (31, 37). Thus, the presence of hCR2 may lead to alterations in B1a B cell function, consistent with the current understanding that B1a cells rely on constant Ag/BCR signaling for positive selection (61, 62). Transgenic mice also develop a relative increase in splenic marginal zone B cells and B-1 cells along with a decrease in follicular B cells. These findings may reflect positive effects of CR2 on marginal zone development, whose numbers and/or function have previously been shown to be deficient in both CD19−/− and Cr2−/− mice (63, 64). Whatever the precise mechanism that underlies this phenotype, the demonstration of decreased numbers of later stage B cells suggests that transgenic B cells do not appropriately progress in development beyond the B220intCD43+CD25− stage, due to either a negative signal or lack of positive selection. These possibilities are under investigation. We also believe that these results provide some insights into why CR2 expression is tightly controlled in both humans and mice and normally first appears only during the IgMhighIgDlow immature cell stage of development (11, 12). Stage-specific expression of CR2 is controlled primarily by an intronic silencer in both the human (47) and mouse (65, 66) genes. Our data demonstrate that premature expression of hCR2 under the control of the Vλ2 promoter/Vλ2–4 enhancer substantially alters B cell development. Thus, CR2 expression may introduce signals into development that reflect local complement activation by foreign or self Ag, something not normally desirous in the pro-B or pre-B cell stages.

Finally, the functional capabilities in response to T-dependent Ag of B cells in hCR2 transgenic mice that do progress through the bone marrow out into the periphery are diminished rather than enhanced. This was demonstrated by the inability to generate a robust switched IgG response to NP-KLH. This diminished response most likely reflects altered B cell selection and the generation of hyporesponsive, or potentially anergic cells. Importantly, in preliminary studies, the decrease in the T-dependent humoral immune response was also found in hCR2int mice (data not shown), strongly suggesting that the decreased response shown in Fig. 6⇑ is not simply due to decreased total IgG levels, but most likely reflects endogenous alterations in transgenic B cells. Also, as noted above, we have shown that hCR2 can act as a coreceptor when cross-linked with the BCR; therefore, hCR2 does not apparently act as a nonfunctional trap or competitive inhibitor of mCR2 for complement-bearing targets. Whatever is the reason for these differences, the data in toto show that marked changes have occurred in transgenic mice that are indicative of alterations of B cell selection. However, further studies are necessary to determine the exact basis for these effects, and whether other responses, such as those to self Ags, would likewise be decreased in transgenic mice.

Acknowledgments

We thank Dr. Karen Francis for help with the immunofluorescence analysis, Lydia Tang for contributions to the ELISA studies, Rachel Henderson for assistance in generating transgenic mice, Karen Helm and Mike Ashton for help with flow cytometry, Roberta Pelanda and Raul Torres for helpful discussions, and Drs. Melanie Hanna and Richard Mead for general assistance.

Footnotes

  • ↵1 These studies were supported by National Institutes of Health Grant R0-1 AI31105 (to V.M.H.), the Wellcome Trust (to B.P.M.), and a Wellcome Trust Prize Traveling Fellowship (to K.J.M.).

  • ↵2 Address correspondence and reprint requests to Dr. V. Michael Holers, Departments of Medicine and Immunology, University of Colorado Health Sciences Center, Box B-115, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: michael.holers{at}uchsc.edu

  • ↵3 Abbreviations used in this paper: BCR, B cell receptor; AP, alkaline phosphatase; CR, complement receptor; hCR, human CR; int, intermediate; KLH, keyhole limpet hemocyanin; m, mouse; NP, nitrophenyl; RT, room temperature; SA, streptavidin; SCR, short consensus repeat; HSA, heat-stable Ag.

  • Received March 14, 2002.
  • Accepted July 25, 2002.

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