IgG1⁺ ovalbumin-specific B-cell transnuclear mice show class switch recombination in rare allelically included B cells

Abstract

We used somatic cell nuclear transfer (SCNT) to generate a mouse from the nucleus of an IgG1⁺ ovalbumin-specific B cell. The resulting OBI mice show generally normal B-cell development, with elevated percentages of marginal zone B cells and a reduction in B-1 B cells. Whereas OBI RAG1^−/− mice have exclusively IgG1 anti-ovalbumin in their serum, OBI mice show elevated levels of anti-ovalbumin of nearly all isotypes 3′ of the γ1 constant region in the IgH locus, indicating that class switch recombination (CSR) occurs in the absence of immunization with ovalbumin. This CSR is associated with the presence of IgM⁺IgG1⁺ double producer B cells that represent <1% of total B cells, accumulate in the peritoneal cavity, and account for near-normal levels of serum IgM and IgG3.

B cells exist as a polyclonal pool such that an antibody response may be mounted against any possible pathogen. When a B-cell recognizes its cognate antigen through its B-cell receptor (BCR), the B cell proliferates and differentiates into antibody-secreting plasma cells and a smaller population of memory B cells. Clonal selection theory rests on the idea that a B cell expresses a BCR of a single specificity; harmful consequences could ensue if a B cell activated in the normal course of an immune response also produced a second antibody that reacted with self-antigen. Several mechanisms ensure that a B cell produces only a single specificity BCR, including monoallelic initiation of recombination, restricted access of the RAG proteins, rapid entry into the cell cycle, chromatin remodeling, and subunit pairing constraints (1, 2). Thus, nearly all B cells express a BCR encoded by single alleles at the IgH and Igκ or λ loci. Allelic exclusion, however, is not perfect, and ∼0.01% of B cells express two rearranged IgH genes (3), whereas 1–7% of B cells express two rearranged Igκ genes (4, 5).

B-cell development begins in the bone marrow when B-cell progenitors express RAG1/2 and rearrange the Ig heavy chain locus (6). D to J rearrangement occurs first, often on both chromosomes, followed by V to DJ rearrangements. A productive, in-frame VDJ results in cell surface expression of the Ig heavy chain paired with VpreB and λ5 surrogate light chains. Pre-BCR signaling induces proliferation and prevents further rearrangements on the other chromosome. After several rounds of cell division, pre-B cells re-express the RAG genes and engage in V to J rearrangement of the Igκ light chain locus. Surface expression of the BCR marks transition to the immature B-cell stage.

Once in the periphery, B cells engage a wider array of antigens, including those from food and commensal microbes. Transitional B cells further differentiate into one of three major B-cell populations: long-lived marginal zone (MZ) B cells that reside in the marginal zone sinus of the spleen; follicular B cells that form the B-cell zones of spleen and lymph nodes; and B-1 B cells that reside mainly in the peritoneal cavity and are a major source of natural antibodies (7, 8). The fate decision made by transitional B cells is linked to the signaling capacity of the BCR, and it has been suggested that BCR affinity for peripheral self-antigens directs B cells into particular lineages, with higher affinity B cells becoming B-1 B cells, whereas lower affinity B cells develop into MZ B cells (7, 8).

When naïve follicular B cells encounter antigen, the BCR-bound antigen complex is internalized into endolysosomes where antigen is degraded and peptide fragments are presented on class II MHC (9). Toll-like receptor (TLR) ligands such as LPS or CpG can further activate a B cell to express the costimulatory ligands CD80 and CD86. Surface expression of peptide-loaded class II MHC with costimulatory ligands engages CD4 T cells of the appropriate specificity. These CD4 T cells provide CD40L stimulation and release cytokines such as IL-4 and IL-6 that induce the formation of a germinal center, where B cells undergo affinity maturation and isotype switching, both of which are mediated by activation-induced deaminase (AID). Rare B cells with mutations that increase their affinity for antigen are selected for expansion and development into plasma cells and memory B cells (9).

All B cells initially express IgM, but may switch to other isotypes upon AID-induced double-strand breaks in the GC-rich switch region that precedes each constant region (10). Resolution of these breaks causes looping out and deletion of the intervening DNA from the genome. Thus, an IgG1⁺ B cell has deleted the μ, δ, and γ3 constant regions, at least on the allele containing the productively rearranged VDJ.

Chicken ovalbumin (OVA), the major protein component of egg whites, has been a favorite of immunologists for years. T-cell receptors from CD8 and CD4 cells specific for immunodominant epitopes of ovalbumin were cloned and used to generate OT-I and OT-II transgenic mice as a source of monoclonal lymphocytes of defined specificity (11, 12). The availability of ovalbumin-specific CD8 and CD4 T cells has inspired the generation of dozens of engineered pathogens, tumor cell lines, and transgenic mice that express ovalbumin to study the many aspects of adaptive immunity. Notwithstanding the widespread use of ovalbumin as a model antigen, no ovalbumin-specific BCR transgenic mice have been reported.

Somatic cell nuclear transfer (SCNT) from antigen-specific lymphocytes allows the generation of transnuclear (TN) mice with lymphocytes of a single, defined specificity (13). Production of TN mice is rapid, requiring ∼6 wk from lymphocyte harvest to obtaining chimeric animals, and requires no DNA vector construction or genetic manipulation of embryonic stem cells. Earlier we reported a panel of TN mice derived from CD8 T cells specific for Toxoplasma gondii (13) and we now applied the same technique to B cells specific for OVA to obtain antigen-specific B-cell transnuclear mice. The resulting OBI mice contain B cells that are ovalbumin specific, have no genetic alterations other than the physiological BCR rearrangements, and are the closest approximation of the high-affinity B cells that result in primary and memory antibody responses in vivo. Our data show that the B cell that served as nucleus donor for SCNT had already class switched to IgG1. Whereas the use of IgG1 is fully compatible with B-cell development, allelic exclusion is imperfect and allows the emergence of B cells that rearrange the remaining wild-type IgH locus to yield a presumably diverse repertoire of IgM. These IgM⁺ IgG1⁺ cells express productively rearranged BCRs of two different specificities and can initiate class-switch recombination in animals not deliberately exposed to ovalbumin, resulting in the production of isotypes other than IgM from the wild-type allele, and ovalbumin-specific class-switched immunoglobulins from the transnuclear allele.

Results

Generation of OBI Mice.

Somatic cell nuclear transfer is most efficient when using F₁ hybrid mice as a source of donor nuclei (13–15). Accordingly we used B6xBALB/c F₁ males as a source of B cells. To identify antigen-specific B cells, we mixed biotinylated ovalbumin with streptavidin-phycoerythrin (PE) to generate tetrameric phycoerythrin-labeled ovalbumin (tOVA-PE). Splenocytes from control mice showed ∼0.03% of B cells binding to tOVA-PE, a frequency too low to proceed with isolation of antigen-specific B cells and SCNT. We therefore immunized mice intraperitoneally with 100 μg of ovalbumin in complete Freund’s adjuvant (CFA), followed by two doses of 100 μg ovalbumin in incomplete Freund’s adjuvant (IFA), which allowed us to identify a rare population (∼0.1%) of B cells that stained with tOVA-PE (Fig. 1A). Seven days after the final immunization, we isolated isotype-switched CD19⁺, IgM⁻, tOVA-PE⁺ B cells by fluorescence activated cell sorting (FACS) and used them as a source of donor nuclei for SCNT. A total of 154 nuclear transfers yielded three ES cell lines, one of which showed tOVA-PE⁺ cells in peripheral blood of chimeric mice and gave germline transmission (Fig. 1B). B cells from the resultant OBI TN mice readily stained with OVA-Alexa 488 and anti-IgG1 (Fig. 1C). The OBI TN IgH and Igκ loci were backcrossed to B6 and placed onto a RAG1^−/− background to prevent endogenous Ig rearrangements. Subsequent experiments were performed on mice that were backcrossed for 8–10 generations onto the B6 or B6;RAG1^−/− backgrounds.

Fig. 1.

OBI mice generated by somatic cell nuclear transfer. B6xBALB/c F₁ male mice were immunized three times with ovalbumin in CFA/IFA adjuvant. Splenocytes were harvested 7 d after the final immunization and stained with anti-IgM and ovalbumin-PE tetramers (tOVA-PE). IgM⁻, ovalbumin⁺ cells were sorted by FACS and used as donor nuclei for SCNT to generate OBI TN mice. (A) Splenocytes from nonimmunized and triply immunized mice were stained with anti-IgM and tOVA-PE. Numbers indicate number of cells per 100,000. (B) Peripheral blood from the chimeric founder shows ovalbumin reactive IgM⁻ B cells. Population shown is gated on CD19⁺ cells. (C) Peripheral blood B cells from germline transmitted OBI mice stain brightly with monomeric ovalbumin-Alexa 488 (Invitrogen) and with anti-IgG1. (D) cDNA with synthesized from OBI RAG1^−/− B cells and subjected to 5′ RACE analysis. The sequence of the IgH and Igκ chains is shown. Nucleotides that differ from germline are highlighted in bold. (E) Overlapping 10-mer peptides from chicken ovalbumin were synthesized and spotted onto nitrocellulose. The membrane was probed with serum from an OBI mouse. Observed reactivity to the peptides is indicated in bold.

B cells sorted from OBI RAG1^−/− mice were used as a source of cDNA for 5′ RACE to determine the sequence of the BCR heavy- and light-chain loci (Fig. 1D), which showed somatic mutations in both the IgH and Igκ variable regions, evidence that the original donor B cell had undergone affinity maturation in a germinal center. The heavy-chain (HC) VDJ was joined to γ1 (IgG1), whereas the light-chain VJ was connected to the κ constant region. Thus, the original donor nucleus came from a high-affinity IgG1⁺Igκ⁺ B cell.

To define the epitope recognized by the OBI BCR, we synthesized overlapping 10-mer peptides from chicken ovalbumin and spotted them onto nitrocellulose. OBI serum recognizes an epitope centered on the sequence DKLPGFGDSI, contained in a surface-exposed loop of ovalbumin (Fig. 1E). The OBI epitope is located in the N-terminal portion of ovalbumin and is distinct from the more C-terminally located OT-I and OT-II epitopes.

OBI Heavy Chain Alone Can Confer Binding to Ovalbumin.

To investigate the role of the OBI heavy chain in antigen binding, we isolated B cells from OBI HC mice that inherited the rearranged OBI heavy chain in the absence of the OBI light chain. OBI HC or wild-type B cells were cultured with CpG for 3 d, labeled to steady state with [³⁵S]methionine/cysteine, and supernatants were immunoprecipitated with ovalbumin-conjugated sepharose beads. Sequential precipitation with ovalbumin-beads removed all anti-ovalbumin antibodies, and the remaining ovalbumin-depleted supernatants were immunoprecipitated with anti-IgM and anti-IgG1 to discern the amount of nonovalbumin-reactive antibodies produced (Fig. 2). Although most of the Ig produced in OBI HC mice is not reactive with ovalbumin, the OBI heavy chain alone is sufficient to confer binding to ovalbumin, when paired with ∼10% (as assessed biochemically) of available light chains.

Fig. 2.

OBI heavy chain alone can confer binding to ovalbumin. B cells were harvested from spleens of B6 mice or mice bearing only the IgH OBI TN locus and cultured for 4 d in media containing LPS. Cells were then labeled with [³⁵S]methionine/cysteine for 4 h. Supernatants were collected and serially immunoprecipitated with ovalbumin-conjugated sepharose beads. After four sequential precipitations, the ova-depleted supernatants were precipitated with protein G to isolate the remaining nonovalbumin reactive antibodies.

OBI Mice Have an Increase in MZ B Cells and a Decrease in B-1 B Cells.

We found no obvious impairment of B-cell development in the OBI mice (Fig. 3). Compared with wild type, OBI and OBI RAG1^−/− bone marrow populations showed a higher percentage of surface BCR⁺ cells, as expected in cells that have already undergone V(D)J recombination (16, 17). The presence of mature B cells in mice that received the OBI heavy chain alone (OBI HC) suggests that IgG1 already pairs adequately with the λ5 surrogate light chain in the course of B-cell development (18). Transgenic light chains have been reported to substitute for λ5 (19); thus in OBI mice with both VDJ and VJ rearrangements, we cannot exclude the possibility that IgG1 pairs with Igκ at the pre–B-cell stage. OBI mice have normal or slightly elevated percentages of B cells in peripheral lymphoid organs, and the absolute numbers of B and T cells in OBI mice are normal.

Fig. 3.

OBI mice have near-normal B-cell development with increased MZ and decreased B-1 B-cell populations. Cells were harvested from spleen, mesenteric lymph nodes, peritoneal cavity, and bone marrow and stained with the indicated antibodies. Spleen populations (Left column) were gated on CD19⁺ cells. Bone marrow populations (Lower Right) were gated on B220⁺ cells. Results are representative of three to six mice per group.

Experiments exploring the role of BCR affinity in fate decisions have thus far relied either on manipulated expression levels of transgenic BCRs and target antigens or on alteration of the signaling capacity of the BCR (7, 8). Here we find a subtle increase in MZ B cells in OBI and OBI RAG1^−/− mice and a decrease in B-1 B cells in OBI mice, which became more pronounced on the RAG1^−/− background (Fig. 3). This skewing of the MZ to B-1 B cell ratio suggests that the particular specificity of the OBI BCR directs cell fate decisions in the transitional B-cell pool.

OBI Serum Antibody Titers Show Extensive Isotype Switching.

In addition to a loss of peritoneal cavity B-1 cells, OBI mice showed fewer CD5⁺ B cells in spleen (Fig. 2). This spleen-resident B-1 B-cell population has been described as composed of short-term antibody-secreting cells and—together with B-1 B cells in the peritoneal cavity—is largely responsible for maintaining serum antibody titers (20, 21). We therefore analyzed serum from 3-mo-old mice of the following genotypes: wild-type B6, OBI HC, OBI, and OBI RAG1^−/− (Fig. 4A). Serum from OBI RAG1^−/− mice contained anti-ovalbumin antibodies composed exclusively of IgG1 and Igκ isotypes, as expected, because OBI RAG1^−/− mice lack the CD4 T cells that provide the CD40L signal to initiate isotype switching (9). However, OBI and even OBI HC mice showed anti-ovalbumin IgG2a, IgG2b, and IgA (Fig. 4A) in the absence of deliberate immunization. To confirm that the secondary antibodies used for ELISA did not cross-react with IgG1, we generated a hybridoma from OBI spleen cells and included hybridoma supernatant in each assay as a positive control for anti-ovalbumin IgG1 and Igκ and a negative control for all other isotypes. The high concentration of anti-ovalbumin IgG1 in OBI serum most likely outcompetes other anti-ovalbumin isotypes for binding to the ovalbumin-coated ELISA plate. Indeed, when serum samples from OB1 mice were first depleted with protein G sepharose to remove IgG and then assayed for ovalbumin-specific IgA, higher titers were detected, which did not plateau over the serum dilutions tested (Fig. 4A, last panel).

Fig. 4.

Serum levels of antiovalbumin antibodies. (A) Serum was harvested from 3-mo-old B6, OBI TN heavy chain only (OBI HC), OBI RAG-proficient (OBI), or OB-I RAG1^−/− mice and analyzed by ELISA for ovalbumin-specific antibodies of the isotypes shown. All mice were born to non-OBI mothers to eliminate the possibility of antiovalbumin antibody transfer via breast milk. Serum was used at a starting concentration of either 100-fold dilution or 1,000-fold dilution (IgG1 and Igκ) and titrated at 10-fold serial dilutions. Error bars indicate SDs of six individual mice per group. Supernatant from an OBI hybridoma (IgG1⁺Igκ⁺) was included on each ELISA plate to control for nonspecific binding of the secondary ELISA reagents to the OBI IgG1. Hybridoma supernatant was used neat and at 10-fold serial dilutions. Results are representative of three independent experiments. Last panel: serum samples were preincubated with protein G sepharose to deplete IgG before analysis by ELISA. (B) Cells were isolated from bone marrow and Peyer’s patches of wild-type, OBI, and OBI RAG1^−/− mice and stained with anti-IgA and fluorescent ovalbumin. Results are representative of two mice per group. Bone marrow populations are gated on CD138⁺ cells. Peyer’s patch populations are gated on CD19⁺ cells.

The B6 mouse genome encodes IgG2c, but not IgG2a (22); therefore, the presence of anti-ovalbumin IgG2a in the OBI mouse strain indicates that it was the BALB/c allele in the original B6xBALB/c F₁ donor B cell that gave rise to the productive VDJ recombination. We could not detect anti-ovalbumin IgG2c in OBI serum, although our detection threshold was higher than for other isotypes, because the anti-IgG2c secondary antibody used for ELISA showed slight reactivity with IgG1 from hybridoma supernatant.

Class-switched serum anti-ovalbumin derives from class-switched plasma cells, so we examined bone marrow from OBI mice. Among the CD138⁺ plasma cells, 6% were IgA⁺ and bound fluorescent ovalbumin (Fig. 4B). The IgA⁺ ovalbumin⁻ plasma cells likely derived from OBI B cells in the germinal center that had sustained AID-induced point mutations that reduced binding to ovalbumin. Because these mice were never immunized, ovalbumin would not be present on follicular dendritic cells, and selection for increased affinity for ovalbumin would not occur. Although the frequency of ovalbumin-reactive IgA⁺ cells was low, none were detected in OBI RAG1^−/− bone marrow. Likewise, Peyer’s patches from OBI mice showed a significant population of IgA⁺ ovalbumin⁺ B cells absent from wild-type and OBI RAG1^−/− mice (Fig. 4B).

Rare Double Producer Cells Account for Isotype Switching in OBI Mice.

How does CSR occur in OBI mice? The lack of CSR in OBI RAG1^−/− mice confirms that CD4 T-cell help is required. However, the frequency of anti-ovalbumin CD4 cells in a naïve polyclonal T-cell pool must be exceedingly small (23). In light of this conundrum, we hypothesized that rare OBI cells might rearrange the wild-type BCR allele and emerge as IgM⁺IgG1⁺ cells. These double producers would have BCRs of at least two different specificities, assuming that allelic exclusion of the light chain locus would be efficient. In such a situation, the non-OBI BCRs might bind to environmental antigens, interact with appropriately specific CD4 T cells, and undergo class switching.

To investigate this possibility, we looked for IgM⁺ cells in OBI mice. Total serum IgM and IgG3 are near-normal in OBI and OBI HC mice (Fig. 5A). However, the number of IgM⁺ cells detectable in spleen was ∼1% (Fig. 5B). Nonetheless, we sorted by FACS IgM⁺IgG1⁺ cells from OBI mice, cultured these cells for 3 d with LPS and CpG, and labeled them with [³⁵S]methionine/cysteine. Immunoprecipitation with anti-κ antibodies recovered equivalent amounts of IgM and IgG1 from the culture media of the double producer cells, demonstrating that they synthesize and secrete both isotypes at comparable efficiencies. These cells are therefore the likely source of serum IgM (Fig. 5C).

Fig. 5.

A rare population of OBI B cells expresses IgM from the endogenous allele and is responsible for maintaining serum IgM levels. (A) Serum was harvested from 3-mo-old male B6, OBI TN heavy chain only (OBI HC), OB-I RAG-proficient (OBI) or OBI RAG1^−/− mice, diluted 100-fold and analyzed by ELISA for total IgM and total IgG3. Three individual mice per group are shown. Error bars show SD of triplicate samples. (B) Splenocytes from an OBI mouse were stained with antibodies to IgM and IgG1. Results are representative of three independent experiments. The IgG1⁺IgM⁺ cells and a comparable number of IgM⁻IgG1⁺ cells were FACS sorted and used in C. (C) IgM⁺IgG1⁺ or IgM⁻IgG1⁺ cells were sorted from OBI spleens by FACS, cultured for 3 d in media containing LPS and CpG. Cells were then labeled with [³⁵S]methionine/cysteine for 4 h. Supernatants were immunoprecipitated with anti-κ antibody to retrieve both IgM and IgG1, and immunoprecipates were digested with endoglycosidase H or endoglycosidase F. *, partially deglycosylated IgM; **, fully deglycosylated IgM or IgG1.

OBI mice have reduced populations of B-1 B cells (Fig. 2). Peritoneal cavity cells from OBI mice were enriched in IgM⁺IgG1⁺ cells, and also IgM⁺IgG1⁻ cells (Fig. 6A). These IgM-only cells, found exclusively in the peritoneal cavity, are either rare cells that failed to express the OBI transnuclear IgG1 and were preferentially directed to and expanded in the B-1 B-cell compartment or arose from IgM⁺IgG1⁺ cells that selectively lost IgG1 expression during affinity maturation (24, 25).

Fig. 6.

Peritoneal cavity is enriched in IgM⁺IgG1⁺ double producers. (A) Peritoneal cavity cells were isolated from wild-type, OBI, and OBI RAG1^−/− mice, stained with antibodies to IgM and IgG1 and analyzed by flow cytometry. Results are representative of three mice per group. (B) Total peritoneal cavity cells were cultured for 3 d in media containing LPS and CpG. Cells were then labeled with [³⁵S]methionine/cysteine for 4 h. Supernatants were immunoprecipitated with anti-κ antibody to retrieve both IgM and IgG1. (C) OBI peritoneal cavity cells were stained with the indicated antibodies and analyzed by flow cytometry. Upper panel is gated on IgG1⁺ cells. Lower Right is gated on CD5⁺IgG1⁺ cells. Results are representative of four individual mice.

Total peritoneal cavity cells were cultured for 3 d with LPS and CpG and labeled with [³⁵S]methionine/cysteine. Immunoprecipitation with anti-κ antibodies recovered a substantial amount of IgM, confirming that the peritoneal cavity is enriched in double producers (Fig. 6B). Of the IgG1⁺ B cells in the peritoneal cavity, 7% also express IgM (Fig. 6C). Of the IgG1⁺CD5⁺ B-1 B-cell population, 81% expressed IgM, suggesting that within the B-1 B-cell compartment, IgM⁺IgG1⁺ cells are the predominant population.

Discussion

Allelic exclusion prevents expression of two different BCRs on a single B cell. Allelic exclusion is mediated in part through suppression of further VDJ recombination once a productively rearranged Ig heavy chain locus has been generated; however, other factors must contribute because two productive VDJ rearrangements can be found in allelically excluded cells (1, 26). We estimate that, similar to IgM⁺ B cells in wild-type mice (3), allelic exclusion applies to 99% of B cells in the OBI mouse as measured by cytofluorimetry. In wild-type mice, cytofluorimetry overestimates the frequency of IgH double expressing cells due to the inclusion of two-cell doublets mis-scored as double producers (3). The actual rate of allelic exclusion in OBI mice is likely to be higher than 99%, although a determination of the exact frequency would require analysis by limiting dilution of sorted putative allelically included cells (3). Despite this tight regulation, a small population of OBI B cells manages to express surface IgM, derived from rearrangement of the wild-type IgH allele. Although such double producers account for <1% of the mature B-cell pool in spleen as estimated by cytofluorimetry, these double-positive B cells, or rather their differentiated progeny, produce enough IgM to maintain near-normal serum IgM levels (27). We cannot exclude the possibility that an environmental antigen cross-reacts with the OBI BCR; however, we view this as unlikely because the vast majority of OBI B cells have a naïve phenotype and OBI mice housed at separate locations have similar levels of the class-switched serum isotypes. Thus, in the absence of deliberate exposure to ovalbumin, double producers are the most likely B cells capable of engaging CD4 T cells. Once AID is activated in a B cell, it acts on transcribed IgH and IgL loci (28), resulting in class switching not only of the wild-type IgH locus (which is the only possible source of IgG3 in OBI mice) but also of the transnuclear OBI IgG1 locus. Thus, OBI mice on a RAG-proficient background express near-normal levels of total serum IgG3 and detectable serum titers of antiovalbumin IgG2a, IgG2b, and IgA.

IgE, the major isotype responsible for asthma and allergies, is generally present at low levels (29). We could not reliably detect antiovalbumin IgE in naïve OBI mice from 6 wk to 6 mo of age. Allergic responses in mice are generally induced by prior sensitization with the antigen. Given that the OBI mice already have a preexpanded pool of antigen-specific B cells and high serum titers of specific antibodies, we were curious as to whether OBI mice would phenocopy ovalbumin-sensitized mice with respect to food allergies. Administration of ovalbumin via the drinking water or by oral gavage of OBI mice did not induce an acute drop in body temperature characteristic of anaphylaxis (30). These results are consistent with the lack of detectable IgE. Nevertheless, if properly sensitized or if induced to class switch to IgE, OBI B cells could be useful for studying allergic responses versus oral tolerance to egg whites, a food that causes significant morbidity in allergic humans.

OBI mice show B-cell subset skewing toward marginal zone B cells with a paucity of B-1 B cells, suggesting either no interaction with self-antigen or a weak signal strength of the OBI BCR (7, 8). This skewed MZ to B-1 B-cell ratio in OBI mice could not be reversed by 4 wk of continuous ovalbumin in the drinking water. Peritoneal cavity (PC) B-1 B cells are capable of T-cell–independent antibody secretion and tend to secrete IgM that reacts weakly with common bacterial cell wall components or with viral constituents (20, 21, 31, 32). The PC B-1 cell compartment, as well as CD5⁺ cells in spleen and lymph nodes, was reduced in OBI mice and absent in OBI RAG1^−/− mice. The few B-1 B cells present in the peritoneal cavity of OBI mice were enriched in IgM⁺IgG1⁺ double producers, with nearly all of the CD5⁺ cells being IgM⁺, suggesting that direction of OBI B cells into the B-1 B-cell lineage is driven largely by the BCR specificities of the allelically included receptor.

IgG1, with its long cytoplasmic tail, differs from IgM in terms of its signaling properties (33, 34). Still, the OBI IgG1 supports B-cell development and mediates nearly complete allelic exclusion in vivo. The OBI heavy chain confers much of the specificity for ovalbumin, and mice bearing only the OBI heavy chain have roughly 10% of the output of their B cells as immunoglobulins specific for ovalbumin, measured by immunoprecipitation. OBI HC mice, which possess polyclonal anti-ovalbumin antibodies in the context of many other B-cell specificities, may more accurately reflect normal physiology and serve as a platform for vaccine development or therapies aimed at activating B-cell memory, especially given that many memory B cells are IgG1⁺ (34).

Here we report a B-cell transnuclear mouse specific for ovalbumin. We defined the epitope, sequenced the rearranged VDJ and VJ loci, and determined that B-cell development and subset formation are, for the most part, normal. OBI B cells secrete anti-ovalbumin antibodies of nearly all isotypes, although isotype switching can be eliminated by crossing to a RAG-deficient background. The OBI mouse will be a valuable resource for studies of antigen-specific B-cell responses.

Materials and Methods

Animal Care.

Animals were housed at the Whitehead Institute for Biomedical Research and maintained according to protocols approved by the Massachusetts Institute of Technology Committee on Animal Care. C57BL/6 and RAG1^−/− mice were purchased from Jackson Labs. TN mice were generated as previously described (13–15).

Sequencing of the BCR Genes.

OBI RAG1^−/− B cells were purified by negative selection using CD43 magnetic beads (Miltenyi Biotec) and used as a source of RNA; 5′RACE was performed according to the manufacturer’s protocol (GeneRacer, L1502-01; Invitrogen).

Flow Cytometry.

Cells from the indicated organs were subjected to hypotonic lysis, stained, and analyzed using a FACSCalibur (BD Pharmingen). Alexa 488 and Alexa 588 ovalbumin were from Invitrogen. All antibodies were from BD Pharmingen.

Serum ELISAs.

Serum was collected from age-matched mice. High-binding 96-well plates (Costar) were coated overnight with 10 mg/mL ovalbumin (Sigma) or 2 μg/mL antimouse H+L (Southern Biotech) diluted in PBS. Plates were washed three times with PBS, 0.05% Tween-20, blocked with 10% (vol/vol) FCS, washed three times, and incubated with serum. Serum samples were used at the indicated dilutions. Hybridoma supernatant was used neat and at 10-fold serial dilutions. Plates were washed five times, and HRP-coupled secondary antibodies recognizing IgM, IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, Igκ, Igλ, or IgE [1 μg/mL in 10% (vol/vol) FCS; Southern Biotech] were added. Plates were washed seven times and bound antibody was detected using 3,3′,5,5′-tetramethylbenzidine substrate.

Production of OBI Hybridoma.

Hybridomas were generated and screened as previously described (35). Spleen cells from OBI HC mice were fused with NS-1 cells. The resulting hybridomas were screened for ovalbumin reactivity by ELISA, and the Igκ genes were amplified by RT-PCR and sequenced. A single OVA-reactive hybridoma bearing the OBI IgG1 and IgVK135 genes was cultured for 3 d in RPMI with low Ig FCS (Gibco), and supernatants were harvested.

Metabolic Labeling and Immunoprecipitation.

B cells were cultured in RPMI with 10% FBS. In some cases, LPS (20 μg/mL) and CpG (1 μM) were added to the culture medium. For metabolic labeling, plasmablasts were starved for 1 h in methionine- and cysteine-free medium, then labeled for 4–6 h with [³⁵S]methionine/cysteine (PerkinElmer). Supernatants were harvested, and cells were lysed in Nonidet P-40 buffer. Supernantants and/or lysates were analyzed by immunoprecipitation, SDS/PAGE, and fluorography. Sequential immunopreciptitations were performed as described (36). Enzymatic deglycosylation was performed using endoglycosidase H (Endo H) or PNGase F (New England Biolabs).