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.