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

Stephanie K. Dougana, Souichi Ogataa,b, Chih-Chi Andrew Hua,c, Gijsbert M. Grotenbrega,d, Eduardo Guillena, Rudolf Jaenischa,1, and Hidde L. Ploegha,1

aWhitehead Institute for Biomedical Research, Cambridge, MA 02142; bJanssen Oncology Research and Development, a division of Janssen Pharmaceutica NV, Beerse B2340, Belgium; cDepartment of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612; and dImmunology Programme and Department of Microbiology, National University of Singapore, Singapore 117456

Contributed by Rudolf Jaenisch, June 22, 2012 (sent for review May 7, 2012)

We used somatic cell nuclear transfer (SCNT) to generate a mouse affinity B cells becoming B-1 B cells, whereas lower affinity B from the nucleus of an IgG1+ ovalbumin-specific B cell. The result- cells develop into MZ B cells (7, 8). ing OBI mice show generally normal B-cell development, with When naïve follicular B cells encounter antigen, the BCR- elevated percentages of marginal zone B cells and a reduction in bound antigen complex is internalized into endolysosomes where B-1 B cells. Whereas OBI RAG1−/− mice have exclusively IgG1 anti- antigen is degraded and peptide fragments are presented on class ovalbumin in their serum, OBI mice show elevated levels of anti- II MHC (9). Toll-like receptor (TLR) ligands such as LPS or CpG ovalbumin of nearly all isotypes 3′ of the γ1 constant region in the can further activate a B cell to express the costimulatory ligands IgH locus, indicating that class switch recombination (CSR) occurs CD80 and CD86. Surface expression of peptide-loaded class II in the absence of immunization with ovalbumin. This CSR is asso- MHC with costimulatory ligands engages CD4 T cells of the ap- propriate specificity. These CD4 T cells provide CD40L stimula- ciated with the presence of IgM+IgG1+ double producer B cells that < tion and release cytokines such as IL-4 and IL-6 that induce the represent 1% of total B cells, accumulate in the peritoneal cavity, formation of a germinal center, where B cells undergo affinity and account for near-normal levels of serum IgM and IgG3. maturation and isotype switching, both of which are mediated by IMMUNOLOGY activation-induced deaminase (AID). Rare B cells with mutations allelic exclusion | natural antibodies | TN mice that increase their affinity for antigen are selected for expansion and development into plasma cells and memory B cells (9). cells exist as a polyclonal pool such that an antibody response All B cells initially express IgM, but may switch to other iso- Bmay be mounted against any possible pathogen. When a B-cell types upon AID-induced double-strand breaks in the GC-rich recognizes its cognate antigen through its B-cell receptor (BCR), switch region that precedes each constant region (10). Resolu- the B cell proliferates and differentiates into antibody-secreting tion of these breaks causes looping out and deletion of the in- plasma cells and a smaller population of memory B cells. Clonal tervening DNA from the genome. Thus, an IgG1+ B cell has selection theory rests on the idea that a B cell expresses a BCR of deleted the μ, δ, and γ3 constant regions, at least on the allele a single specificity; harmful consequences could ensue if a B cell containing the productively rearranged VDJ. activated in the normal course of an immune response also pro- Chicken ovalbumin (OVA), the major protein component of duced a second antibody that reacted with self-antigen. Several egg whites, has been a favorite of immunologists for years. T-cell mechanisms ensure that a B cell produces only a single specificity receptors from CD8 and CD4 cells specific for immunodominant BCR, including monoallelic initiation of recombination, restricted epitopes of ovalbumin were cloned and used to generate OT-I access of the RAG proteins, rapid entry into the cell cycle, chro- and OT-II transgenic mice as a source of monoclonal lympho- matin remodeling, and subunit pairing constraints (1, 2). Thus, cytes of defined specificity (11, 12). The availability of ovalbu- nearly all B cells express a BCR encoded by single alleles at the IgH min-specific CD8 and CD4 T cells has inspired the generation and Igκ or λ loci. Allelic exclusion, however, is not perfect, and of dozens of engineered pathogens, tumor cell lines, and trans- ∼0.01% of B cells express two rearranged IgH genes (3), whereas genic mice that express ovalbumin to study the many aspects of 1–7% of B cells express two rearranged Igκ genes (4, 5). adaptive immunity. Notwithstanding the widespread use of ov- B-cell development begins in the bone marrow when B-cell albumin as a model antigen, no ovalbumin-specific BCR trans- progenitors express RAG1/2 and rearrange the Ig heavy chain genic mice have been reported. locus (6). D to J rearrangement occurs first, often on both chro- Somatic cell nuclear transfer (SCNT) from antigen-specific mosomes, followed by V to DJ rearrangements. A productive, in- lymphocytes allows the generation of transnuclear (TN) mice frame VDJ results in cell surface expression of the Ig heavy chain with lymphocytes of a single, defined specificity (13). Production paired with VpreB and λ5 surrogate light chains. Pre-BCR sig- of TN mice is rapid, requiring ∼6 wk from lymphocyte harvest to naling induces proliferation and prevents further rearrangements obtaining chimeric animals, and requires no DNA vector con- on the other chromosome. After several rounds of cell division, struction or genetic manipulation of embryonic stem cells. Earlier pre-B cells re-express the RAG genes and engage in V to J rear- we reported a panel of TN mice derived from CD8 T cells specific rangement of the Igκ light chain locus. Surface expression of the for Toxoplasma gondii (13) and we now applied the same tech- BCR marks transition to the immature B-cell stage. nique to B cells specific for OVA to obtain antigen-specific B-cell Once in the periphery, B cells engage a wider array of anti- transnuclear mice. The resulting OBI mice contain B cells that gens, including those from food and commensal microbes. are ovalbumin specific, have no genetic alterations other than the 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 Author contributions: S.K.D., S.O., R.J., and H.L.P. designed research; S.K.D. and S.O. that form the B-cell zones of spleen and lymph nodes; and B-1 B performed research; G.M.G. and E.G. contributed new reagents/analytic tools; S.K.D., cells that reside mainly in the peritoneal cavity and are a major C.-C.A.H., R.J., and H.L.P. analyzed data; and S.K.D. and H.L.P. wrote the paper. source of natural antibodies (7, 8). The fate decision made by The authors declare no conflict of interest. transitional B cells is linked to the signaling capacity of the BCR, Freely available online through the PNAS open access option. and it has been suggested that BCR affinity for peripheral self- 1To whom correspondence may be addressed. E-mail: [email protected] or jaenisch@wi. antigens directs B cells into particular lineages, with higher mit.edu.

www.pnas.org/cgi/doi/10.1073/pnas.1210273109 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 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 tet- rameric 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-specificB 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 chi- meric 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 gen- − − erations onto the B6 or B6;RAG1 / backgrounds. − − 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 con- stant 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 syn- thesized overlapping 10-mer peptides from chicken ovalbumin and spotted them onto nitrocellulose. OBI serum recognizes an epitope centered on the sequence DKLPGFGDSI, contained in Fig. 1. OBI mice generated by somatic cell nuclear transfer. B6xBALB/c F1 a surface-exposed loop of ovalbumin (Fig. 1E). The OBI epitope male mice were immunized three times with ovalbumin in CFA/IFA adjuvant. is located in the N-terminal portion of ovalbumin and is distinct Splenocytes were harvested 7 d after the final immunization and stained with − from the more C-terminally located OT-I and OT-II epitopes. 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) OBI Heavy Chain Alone Can Confer Binding to Ovalbumin. To in- Splenocytes from nonimmunized and triply immunized mice were stained with anti-IgM and tOVA-PE. Numbers indicate number of cells per 100,000. (B)Pe- vestigate the role of the OBI heavy chain in antigen binding, we − ripheral blood from the chimeric founder shows ovalbumin reactive IgM isolated B cells from OBI HC mice that inherited the rearranged B cells. Population shown is gated on CD19+ cells. (C) Peripheral blood B cells OBI heavy chain in the absence of the OBI light chain. OBI HC from germline transmitted OBI mice stain brightly with monomeric ovalbumin- or wild-type B cells were cultured with CpG for 3 d, labeled to 35 Alexa 488 (Invitrogen) and with anti-IgG1. (D) cDNA with synthesized from steady state with [ S]methionine/cysteine, and supernatants OBI RAG1−/− Bcellsandsubjectedto5′ RACE analysis. The sequence of the IgH were immunoprecipitated with ovalbumin-conjugated sepharose 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 wild type cell lysate OBI heavy chain cell lysate wild type media OBI heavy chain media an OBI mouse. Observed reactivity to the peptides is indicated in bold. post post post post total Ova total Ova total Ova total Ova Ig Ova1 Ova2 Ova3 Ig Ig Ova1 Ova2 Ova3 Ig Ig Ova1 Ova2 Ova3 Ig Ig Ova1 Ova2 Ova3 Ig

150 physiological BCR rearrangements, and are the closest approx- imation of the high-affinity B cells that result in primary and 100

memory antibody responses in vivo. Our data show that the B 75 cell that served as nucleus donor for SCNT had already class IgM

switched to IgG1. Whereas the use of IgG1 is fully compatible 50 IgG1 with B-cell development, allelic exclusion is imperfect and allows

the emergence of B cells that rearrange the remaining wild-type 37 IgH locus to yield a presumably diverse repertoire of IgM. These IgM+ IgG1+ cells express productively rearranged BCRs of two 25 Ig 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 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 transnuclear allele. 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 immu- Results noprecipitated with ovalbumin-conjugated sepharose beads. After four se- Generation of OBI Mice. Somatic cell nuclear transfer is most effi- quential precipitations, the ova-depleted supernatants were precipitated cient when using F1 hybrid mice as a source of donor nuclei (13– with protein G to isolate the remaining nonovalbumin reactive antibodies.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1210273109 Dougan et al. Downloaded by guest on October 2, 2021 beads. Sequential precipitation with ovalbumin-beads removed 1.8 IgG1 1.8 Igκ 1.8 Igλ A 1.6 1.6 1.6 all anti-ovalbumin antibodies, and the remaining ovalbumin- 1.4 1.4 1.4 OBI HC 1.2 1.2 1.2 OBI depleted supernatants were immunoprecipitated with anti-IgM 1 1 1 OBI RAG1-/- 0.8 0.8 0.8 wild type and anti-IgG1 to discern the amount of nonovalbumin-reactive 0.6 0.6 0.6 hybridoma anti-ova O.D. anti-ova anti-ova O.D. anti-ova 0.4 0.4 O.D. anti-ova 0.4 antibodies produced (Fig. 2). Although most of the Ig produced in 0.2 0.2 0.2 0 0 0 -6 -4 -3 -6 -4 -3 -3 -2 OBI HC mice is not reactive with ovalbumin, the OBI heavy chain 10 10-5 10 10 10 10-5 10 10 10-5 10-4 10 10 alone is sufficient to confer binding to ovalbumin, when paired 1.8 IgG2a 1.8 IgG2b 1.8 IgA 1.8 IgA (IgG-depleted) with ∼10% (as assessed biochemically) of available light chains. 1.6 1.6 1.6 1.6 1.4 1.4 1.4 1.4 1.2 1.2 1.2 1.2 1 1 1 1 OBI Mice Have an Increase in MZ B Cells and a Decrease in B-1 B Cells. 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 anti-ova O.D. anti-ova anti-ova O.D. anti-ova anti-ova O.D. anti-ova We found no obvious impairment of B-cell development in the O.D. anti-ova 0.4 0.4 0.4 0.4 OBI mice (Fig. 3). Compared with wild type, OBI and OBI 0.2 0.2 0.2 0.2 − − 0 0 0 0 / -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 RAG1 bone marrow populations showed a higher percentage 10-5 10-4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 + of surface BCR cells, as expected in cells that have already wild type OBI OBI RAG1-/-

B (gated on CD138+) undergone V(D)J recombination (16, 17). The presence of ma- 0.6% 6.2% 0.0% ture B cells in mice that received the OBI heavy chain alone Bone marrow (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 36% 22% 0.0% (19); thus in OBI mice with both VDJ and VJ rearrangements,

κ (gated on CD19+) we cannot exclude the possibility that IgG1 pairs with Ig at the 0.28% 0.69% 0.01% patches Peyer’s –

pre B-cell stage. OBI mice have normal or slightly elevated OVA-Alexa555 percentages of B cells in peripheral lymphoid organs, and the absolute numbers of B and T cells in OBI mice are normal. Experiments exploring the role of BCR affinity in fate decisions 2.6% 4.2% 0.0% have thus far relied either on manipulated expression levels of transgenic BCRs and target antigens or on alteration of the sig- IgA naling capacity of the BCR (7, 8). Here we find a subtle increase −/− Fig. 4. Serum levels of antiovalbumin antibodies. (A) Serum was harvested in MZ B cells in OBI and OBI RAG1 mice and a decrease in from 3-mo-old B6, OBI TN heavy chain only (OBI HC), OBI RAG-proficient IMMUNOLOGY B-1 B cells in OBI mice, which became more pronounced on the (OBI), or OB-I RAG1−/− mice and analyzed by ELISA for ovalbumin-specific −/− RAG1 background (Fig. 3). This skewing of the MZ to B-1 B antibodies of the isotypes shown. All mice were born to non-OBI mothers to cell ratio suggests that the particular specificity of the OBI BCR eliminate the possibility of antiovalbumin antibody transfer via breast milk. directs cell fate decisions in the transitional B-cell pool. 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. OBI Serum Antibody Titers Show Extensive Isotype Switching. In Error bars indicate SDs of six individual mice per group. Supernatant from an addition to a loss of peritoneal cavity B-1 cells, OBI mice showed OBI hybridoma (IgG1+Igκ+) was included on each ELISA plate to control for fewer CD5+ B cells in spleen (Fig. 2). This spleen-resident B-1 nonspecific binding of the secondary ELISA reagents to the OBI IgG1. Hy- B-cell population has been described as composed of short-term bridoma supernatant was used neat and at 10-fold serial dilutions. Results antibody-secreting cells and—together with B-1 B cells in the are representative of three independent experiments. Last panel: serum peritoneal cavity—is largely responsible for maintaining 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 antibody titers (20, 21). We therefore analyzed serum from 3- − − patches of wild-type, OBI, and OBI RAG1 / mice and stained with anti-IgA mo-old mice of the following genotypes: wild-type B6, OBI HC, 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. wild type OBI RAG1+/- OBI RAG1-/- wild type OBI RAG1+/- OBI RAG1-/- yp oe Peritoneal cavity Lymph nodes 8.7 10 − − − − 18 59 45 1 / A / 37 51 98 OBI, and OBI RAG1 (Fig. 4 ). Serum from OBI RAG1 75 65 CD3 CD21 68 mice contained anti-ovalbumin antibodies composed exclusively − − of IgG1 and Igκ isotypes, as expected, because OBI RAG1 / CD23 B220 mice lack the CD4 T cells that provide the CD40L signal to 2.6 1.4 0.12 17 4.2 2.2 initiate isotype switching (9). However, OBI and even OBI HC mice showed anti-ovalbumin IgG2a, IgG2b, and IgA (Fig. 4A)in CD5 68 CD19 26 32 the absence of deliberate immunization. To confirm that the B220 B220 secondary antibodies used for ELISA did not cross-react with

spleen IgG1, we generated a hybridoma from OBI spleen cells and in- 16 16 11 21 922 10 16 18 cluded hybridoma supernatant in each assay as a positive control 67 45 45 for anti-ovalbumin IgG1 and Igκ and a negative control for all Bone marrow AA4.1 CD43 15 32 32 other isotypes. The high concentration of anti-ovalbumin IgG1 in CD23 CD19 OBI serum most likely outcompetes other anti-ovalbumin iso- types for binding to the ovalbumin-coated ELISA plate. Indeed, 7 39 36 when serum samples from OB1 mice were first depleted with 2

B220 3.0 1.2 1.3 protein G sepharose to remove IgG and then assayed for oval- 11 fi IgM or IgG1 bumin-speci c IgA, higher titers were detected, which did not GL7 IgD plateau over the serum dilutions tested (Fig. 4A, last panel). The B6 mouse genome encodes IgG2c, but not IgG2a (22); 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, therefore, the presence of anti-ovalbumin IgG2a in the OBI mesenteric lymph nodes, peritoneal cavity, and bone marrow and stained mouse strain indicates that it was the BALB/c allele in the with the indicated antibodies. Spleen populations (Left column) were gated original B6xBALB/c F1 donor B cell that gave rise to the pro- on CD19+ cells. Bone marrow populations (Lower Right) were gated on ductive VDJ recombination. We could not detect anti-ovalbumin B220+ cells. Results are representative of three to six mice per group. IgG2c in OBI serum, although our detection threshold was

Dougan et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 higher than for other isotypes, because the anti-IgG2c secondary A 200 200 B 1.0% antibody used for ELISA showed slight reactivity with IgG1 from

L) 150 150 m g/mL)

hybridoma supernatant. g/ μ μ

Class-switched serum anti-ovalbumin derives from class-switched 100 100 IgM um IgM ( plasma cells, so we examined bone marrow from OBI mice. Among r 50 50 se the CD138+ plasma cells, 6% were IgA+ and bound fluorescent serum IgG3 ( + − 0 0 ovalbumin (Fig. 4B). The IgA ovalbumin plasma cells likely wild OBI HC OBI OBI wild OBI HC OBI OBI derived from OBI B cells in the germinal center that had sustained type RAG1-/- type RAG1-/- IgG1 AID-induced point mutations that reduced binding to ovalbumin. Because these mice were never immunized, ovalbumin would not C Supernatant/medium be present on follicular dendritic cells, and selection for increased IgM (+), IgG1(+) IgM (-), IgG1(+) Unsorted affinity for ovalbumin would not occur. Although the frequency of - EndoH EndoF --EndoH EndoF EndoH EndoF + ovalbumin-reactive IgA cells was low, none were detected in OBI 150 − − RAG1 / bone marrow. Likewise, Peyer’s patches from OBI mice showed a significant population of IgA+ ovalbumin+ B cells absent 100 − − from wild-type and OBI RAG1 / mice (Fig. 4B). 75 IgM Rare Double Producer Cells Account for Isotype Switching in OBI Mice. IgM* How does CSR occur in OBI mice? The lack of CSR in OBI IgM** −/− fi RAG1 mice con rms that CD4 T-cell help is required. 50 IgG1 However, the frequency of anti-ovalbumin CD4 cells in a naïve IgG1** 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+ 37 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 ap- fi propriately speci c CD4 T cells, and undergo class switching. Igκ To investigate this possibility, we looked for IgM+ cells in OBI 25 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/ Fig. 5. A rare population of OBI B cells expresses IgM from the endogenous cysteine. Immunoprecipitation with anti-κ antibodies recovered 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 equivalent amounts of IgM and IgG1 from the culture media of −/− RAG-proficient (OBI) or OBI RAG1 mice, diluted 100-fold and analyzed by the double producer cells, demonstrating that they synthesize and fi ELISA for total IgM and total IgG3. Three individual mice per group are secrete both isotypes at comparable ef ciencies. These cells are shown. Error bars show SD of triplicate samples. (B) Splenocytes from an therefore the likely source of serum IgM (Fig. 5C). OBI mouse were stained with antibodies to IgM and IgG1. Results are OBI mice have reduced populations of B-1 B cells (Fig. 2). representative of three independent experiments. The IgG1+IgM+ cells Peritoneal cavity cells from OBI mice were enriched in and a comparable number of IgM−IgG1+ cells were FACS sorted and used in + + + − IgM IgG1 cells, and also IgM IgG1 cells (Fig. 6A). These C.(C)IgM+IgG1+ or IgM−IgG1+ cells were sorted from OBI spleens by FACS, IgM-only cells, found exclusively in the peritoneal cavity, are cultured for 3 d in media containing LPS and CpG. Cells were then labeled either rare cells that failed to express the OBI transnuclear IgG1 with [35S]methionine/cysteine for 4 h. Supernatants were immunoprecipi- and were preferentially directed to and expanded in the B-1 B- tated with anti-κ antibody to retrieve both IgM and IgG1, and immuno- cell compartment or arose from IgM+IgG1+ cells that selectively precipates were digested with endoglycosidase H or endoglycosidase F. *, lost IgG1 expression during affinity maturation (24, 25). partially deglycosylated IgM; **, fully deglycosylated IgM or IgG1. Total peritoneal cavity cells were cultured for 3 d with LPS and CpG and labeled with [35S]methionine/cysteine. Immu- noprecipitation with anti-κ antibodies recovered a substan- likely to be higher than 99%, although a determination of the tial amount of IgM, confirming that the peritoneal cavity is exact frequency would require analysis by limiting dilution of enriched in double producers (Fig. 6B). Of the IgG1+ B cells sorted putative allelically included cells (3). Despite this tight in the peritoneal cavity, 7% also express IgM (Fig. 6C). Of the regulation, a small population of OBI B cells manages to express IgG1+CD5+ B-1 B-cell population, 81% expressed IgM, sug- surface IgM, derived from rearrangement of the wild-type IgH gesting that within the B-1 B-cell compartment, IgM+IgG1+ cells allele. Although such double producers account for <1% of the are the predominant population. mature B-cell pool in spleen as estimated by cytofluorimetry, these double-positive B cells, or rather their differentiated Discussion progeny, produce enough IgM to maintain near-normal serum Allelic exclusion prevents expression of two different BCRs on IgM levels (27). We cannot exclude the possibility that an envi- a single B cell. Allelic exclusion is mediated in part through ronmental antigen cross-reacts with the OBI BCR; however, we suppression of further VDJ recombination once a productively view this as unlikely because the vast majority of OBI B cells rearranged Ig heavy chain locus has been generated; however, have a naïve phenotype and OBI mice housed at separate lo- other factors must contribute because two productive VDJ cations have similar levels of the class-switched serum isotypes. rearrangements can be found in allelically excluded cells (1, 26). Thus, in the absence of deliberate exposure to ovalbumin, double We estimate that, similar to IgM+ B cells in wild-type mice (3), producers are the most likely B cells capable of engaging CD4 T allelic exclusion applies to 99% of B cells in the OBI mouse as cells. Once AID is activated in a B cell, it acts on transcribed IgH measured by cytofluorimetry. In wild-type mice, cytofluorimetry and IgL loci (28), resulting in class switching not only of the wild- overestimates the frequency of IgH double expressing cells due type IgH locus (which is the only possible source of IgG3 in OBI to the inclusion of two-cell doublets mis-scored as double pro- mice) but also of the transnuclear OBI IgG1 locus. Thus, OBI ducers (3). The actual rate of allelic exclusion in OBI mice is mice on a RAG-proficient background express near-normal

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1210273109 Dougan et al. Downloaded by guest on October 2, 2021 CD5+ cells being IgM+, suggesting that direction of OBI B cells A wild type OBI OBI RAG1-/- into the B-1 B-cell lineage is driven largely by the BCR specif- 28 0.0 1.5 2.1 0.0 0.0 icities 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 IgM 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 IgG1 roughly 10% of the output of their B cells as immunoglobulins specific for ovalbumin, measured by immunoprecipitation. OBI B C OBI 150 HC mice, which possess polyclonal anti-ovalbumin antibodies in 7 the context of many other B-cell specificities, may more accurately 100 reflect normal physiology and serve as a platform for vaccine de- 75 IgM velopment or therapies aimed at activating B-cell memory, espe- IgM cially given that many memory B cells are IgG1+ (34). 93 Here we report a B-cell transnuclear mouse specific for oval- 50 fi IgG1 bumin. We de ned the epitope, sequenced the rearranged VDJ IgG1 and VJ loci, and determined that B-cell development and subset formation are, for the most part, normal. OBI B cells secrete 37 anti-ovalbumin antibodies of nearly all isotypes, although isotype 81 switching can be eliminated by crossing to a RAG-deficient background. The OBI mouse will be a valuable resource for 1.4%

IgM fi

CD5 studies of antigen-speci c B-cell responses.

25 Igκ 19 Materials and Methods Animal Care. Animals were housed at the Whitehead Institute for Biomedical IgG1 IgG1 Research and maintained according to protocols approved by the Massa-

+ + chusetts Institute of Technology Committee on Animal Care. C57BL/6 and Fig. 6. Peritoneal cavity is enriched in IgM IgG1 double producers. (A) −/−

− − RAG1 mice were purchased from Jackson Labs. TN mice were generated IMMUNOLOGY Peritoneal cavity cells were isolated from wild-type, OBI, and OBI RAG1 / as previously described (13–15). mice, stained with antibodies to IgM and IgG1 and analyzed by flow cytometry. Results are representative of three mice per group. (B) Total Sequencing of the BCR Genes. OBI RAG1−/− B cells were purified by negative peritoneal cavity cells were cultured for 3 d in media containing LPS and selection using CD43 magnetic beads (Miltenyi Biotec) and used as a source CpG. Cells were then labeled with [35S]methionine/cysteine for 4 h. Super- of RNA; 5′RACE was performed according to the manufacturer’s protocol natants were immunoprecipitated with anti-κ antibody to retrieve both IgM (GeneRacer, L1502-01; Invitrogen). 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 Flow Cytometry. Cells from the indicated organs were subjected to hypotonic four individual mice. lysis, stained, and analyzed using a FACSCalibur (BD Pharmingen). Alexa 488 and Alexa 588 ovalbumin were from Invitrogen. All antibodies were from BD Pharmingen. levels of total serum IgG3 and detectable serum titers of anti- ovalbumin IgG2a, IgG2b, and IgA. Serum ELISAs. Serum was collected from age-matched mice. High-binding 96- IgE, the major isotype responsible for asthma and allergies, is 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 generally present at low levels (29). We could not reliably detect washed three times with PBS, 0.05% Tween-20, blocked with 10% (vol/vol) antiovalbumin IgE in naïve OBI mice from 6 wk to 6 mo of age. FCS, washed three times, and incubated with serum. Serum samples were Allergic responses in mice are generally induced by prior sensi- used at the indicated dilutions. Hybridoma supernatant was used neat and tization with the antigen. Given that the OBI mice already have at 10-fold serial dilutions. Plates were washed five times, and HRP-coupled a preexpanded pool of antigen-specific B cells and high serum secondary antibodies recognizing IgM, IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, titers of specific antibodies, we were curious as to whether OBI Igκ,Igλ,orIgE[1μg/mL in 10% (vol/vol) FCS; Southern Biotech] were added. mice would phenocopy ovalbumin-sensitized mice with respect Plates were washed seven times and bound antibody was detected using to food allergies. Administration of ovalbumin via the drinking 3,3′,5,5′-tetramethylbenzidine substrate. water or by oral gavage of OBI mice did not induce an acute drop in body temperature characteristic of anaphylaxis (30). Production of OBI Hybridoma. Hybridomas were generated and screened as These results are consistent with the lack of detectable IgE. previously described (35). Spleen cells from OBI HC mice were fused with NS-1 Nevertheless, if properly sensitized or if induced to class switch cells. The resulting hybridomas were screened for ovalbumin reactivity by ELISA, to IgE, OBI B cells could be useful for studying allergic responses and the Igκ genes were amplified by RT-PCR and sequenced. A single OVA- versus oral tolerance to egg whites, a food that causes significant reactive hybridoma bearing the OBI IgG1 and IgVK135 genes was cultured for morbidity in allergic humans. 3 d in RPMI with low Ig FCS (Gibco), and supernatants were harvested. OBI mice show B-cell subset skewing toward marginal zone B cells with a paucity of B-1 B cells, suggesting either no in- Metabolic Labeling and Immunoprecipitation. B cells were cultured in RPMI μ μ teraction with self-antigen or a weak signal strength of the OBI with 10% FBS. In some cases, LPS (20 g/mL) and CpG (1 M) were added to the culture medium. For metabolic labeling, plasmablasts were starved for 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 1 h in methionine- and cysteine-free medium, then labeled for 4 6 h with [35S]methionine/cysteine (PerkinElmer). Supernatants were harvested, and drinking water. Peritoneal cavity (PC) B-1 B cells are capable of – cells were lysed in Nonidet P-40 buffer. Supernantants and/or lysates were T-cell independent antibody secretion and tend to secrete IgM analyzed by immunoprecipitation, SDS/PAGE, and fluorography. Sequential that reacts weakly with common bacterial cell wall components immunopreciptitations were performed as described (36). Enzymatic degly- or with viral constituents (20, 21, 31, 32). The PC B-1 cell cosylation was performed using endoglycosidase H (Endo H) or PNGase F compartment, as well as CD5+ cells in spleen and lymph nodes, − − (New England Biolabs). 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 ACKNOWLEDGMENTS. We thank Patti Wisniewski and Chad Araneo for + + enriched in IgM IgG1 double producers, with nearly all of the cell sorting and John Jackson for mouse husbandry. Oktay Kirak performed

Dougan et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 SCNT and the epitope mapping experiments. S.K.D. received a fellowship ceutica NV. H.L.P. and R.J. are funded by grants from the National from the Cancer Research Institute. S.O. was funded by Janssen Pharma- Institutes of Health.

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