VH replacement in primary immunoglobulin repertoire diversification Amy Suna,1, Tatiana I. Novobrantsevab,1,2, Maryaline Coffrea, Susannah L. Hewitta, Kari Jensenb, Jane A. Skoka, Klaus Rajewskyb,3, and Sergei B. Koralova,4 a

Department of Pathology, New York University School of Medicine, New York, NY 10016; and bImmune Disease Institute, Boston, MA 02115

Edited by Frederick W. Alt, Howard Hughes Medical Institute, Boston Children’s Hospital and Harvard Medical School, Boston, MA, and approved December 19, 2014 (received for review September 18, 2014)

The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAGmediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of selfreactive antibodies.

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VH replacement receptor editing secondary rearrangement V(D)J

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occur. This progression is accompanied by a burst of proliferation, which ensures that the functional IgH rearrangement is not lost in the event of unsuccessful IgL recombination and increases diversity by allowing a given IgH chain to pair with multiple IgL chains. Only cells that acquire a functional B-cell receptor (BCR), with an in-frame rearranged IgH chain that successfully pairs with a productively rearranged IgL chain, progress to become mature B cells. The imprecise nature of the joining process in V(D)J recombination contributes to the diversity of the antibody repertoire, but also leads to a significant number of nonfunctional rearrangements, with approximately two-thirds of the joints being out-of-frame. At the IgH locus, VH replacement can rescue “dead-end” pro-B cells that have acquired nonproductive IgH joints on both alleles by rearranging an upstream VH gene with a nonfunctional VHDHJH (4–7). Ordered rearrangement of the IgH and IgL loci is mediated by the RAG1/2 proteins that recognize RSSs flanking V, D, and J segments. The consensus RSS is composed of a heptamer (CACTGTG) and a nonamer (GGTTTTTGT) separated by either a 12-bp or 23-bp spacer (8). V(D)J recombination occurs preferentially between gene segments flanked by RSSs of dissimilar lengths, thus directing the order of recombination; this is known as the 12/23 rule. Because the VHDHJH recombination process eliminates any remaining DH genes and their flanking 12-bp RSSs, further editing of this locus requires a recombination Significance

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The recombinatorial process of V(D)J rearrangement generates a vast antibody repertoire from a limited number of genes. The joints generated in the course of V(D)J recombination are imprecise thus yielding greater diversity but also resulting in frequent generation of nonproductive VDJ rearrangements. We have previously shown that B cells with two nonproductive IgH rearrangements can be efficiently rescued by a form of secondary V(D)J recombination called VH replacement. We now demonstrate that VH replacement also contributes to the diversity of the immune repertoire by modifying productive IgH rearrangements. Results presented herein suggest that VH replacement occurs exclusively during early stages of B-cell development and therefore does not contribute to the editing of self-reactive antibodies.

A

hallmark of the adaptive immune system is its ability to generate a large antibody repertoire despite a limited genome through the rearrangement of variable (V), diversity (D), and joining (J) genes during B-cell development in a process called V(D)J recombination (1). Each antibody-producing cell expresses a single pair of heavy and light chains generated by this process during the pro–B-cell and pre–B-cell stages of development, respectively. V(D)J recombination is mediated by the recombination activating proteins RAG1 and RAG2, which bind and cleave recombination recognition sequences (RSSs) flanking V, D, and J genes. Because there are several members of V, D (for the heavy chain), and J segments, the combinatorial nature of V(D)J rearrangement allows for the generation of a vastly diverse antibody repertoire from a relatively modest amount of genetic information present in the germline DNA. Ig gene rearrangement progresses in an ordered stepwise manner during B-cell development, with Ig heavy (IgH) chain assembly before Ig light (IgL) chain assembly. In-frame rearrangement of a VHDHJH joint leads to expression of an IgH chain that pairs with an invariant surrogate light chain, and, in association with the Igα and Igβ signal-transducing subunits, these proteins form the pre–B-cell receptor (pre-BCR) (2, 3). Signaling through a functional pre-BCR allows the cell to progress from the pro–Bcell stage to the pre–B-cell stage, where IgL rearrangement can E458–E466 | PNAS | Published online January 21, 2015

Author contributions: A.S., T.I.N., J.A.S., K.R., and S.B.K. designed research; A.S., T.I.N., M.C., S.L.H., K.J., J.A.S., and S.B.K. performed research; A.S., T.I.N., and S.B.K. analyzed data; and A.S., K.R., and S.B.K. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

A.S. and T.I.N. contributed equally to this work.

2

Present address: Jounce Therapeutics, Cambridge, MA 02138.

3

Present address: Max Delbrück Center for Molecular Medicine, Berlin, Germany 13092.

4

To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1418001112/-/DCSupplemental.

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Results Generation of a Mouse Strain with an IgH Locus Containing a Functional VHDHJH Rearrangement. Using a gene targeting and screening strat-

egy similar to that described previously (4), we generated a novel transgenic mouse with an IgH locus that mimics a physiologically recombined VHDHJH allele, called D23prod, encoding the IgH chain of a polyreactive antibody, D23 (Fig. 1A). This antibody, expressed by the D23 hybridoma, has a broad range of specificities, including ssDNA and dsDNA (13). The Q52.2.4 VH gene of the D23 IgH chain encodes a well-conserved cRSS toward the 3′ end of the VH gene, and previous studies, including our own, have demonstrated VH replacement of VHDHJH joints containing the Q52 family VH genes (4, 6, 7). We knocked the D23 IgH rearrangement into the IgH locus in 129 IB10 ES cells, preserving all VH elements upstream of VH81X while deleting all downstream DH and JH elements, thereby generating a VHDHJH knock-in that faithfully reproduces an endogenous productive VHDHJH rearrangement (Fig. 1A). The VHDHJH was introduced in the JH gene region, whereas a double loxP site was knocked into the region upstream of the VH81X gene, the most 3′ VH element. Subsequent Cre-mediated recombination between the upstream double loxP and the loxP flanking the introduced VDJ rearrangement resulted in the deletion of the intervening DH elements. This is the same strategy used to Sun et al.

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generate our previously published mouse model of VH replacement, D23stop, so named because of a stop codon introduced in the D23 VH gene (4). Whereas we originally eliminated the downstream JH genes to readily distinguish between the new D23prod allele and the nonproductive D23stop allele, the lack of JH elements also precludes rare VH-to-JH rearrangements that we previously observed in B cells harboring two D23stop alleles (4, 14). Mice Heterozygous for D23prod Have a Reduced Pro–B-Cell Compartment.

Analysis of the bone marrow of mice heterozygous for the D23prod allele (D23prod/+ ) revealed a reduced compartment of progenitor B lymphocytes (B220+ IgM− ) compared with bone marrow from WT mice (Fig. 1B). Examination of this subset demonstrated a dramatic reduction in pro-B cells (c-kit+ CD25−), with a concomitant increase in the pre–B-cell compartment (c-kit− CD25+). Because D23prod progenitor B cells enter the pro–B-cell compartment with an in-frame VHDHJH rearrangement, the transit of these cells through this stage of development likely is accelerated. There was no difference in the proportions of recirculating (B220hi IgM+) and immature (B220lo IgM+) B cells between mutants and controls. In the periphery, we observed a substantial accumulation of cells in the marginal zone compartment of D23prod/+ mice (Fig. 1C; CD1dhi CD23lo), whereas the number of follicular B cells (B220+ CD23hi) was normal. This analysis reveals that despite accelerated B-cell development, peripheral B-cell compartments appear mostly normal in D23prod/+ mice. Evidence of Inactivation of the D23prod IgH Allele. The D23prod

VHDHJH targeting was performed in IB10 ES cells from the 129/ola background; thus, mice homozygous for the D23prod allele expressed IgM of the a allotype on the surface of B cells. We crossed the D23prod allele onto the C56Bl/6 background, where the expressed IgM is of the b allotype. To monitor use of the WT IgH allele or D23prod IgH allele in D23prod/+ animals, we used cell surface antibodies specific for the IgM a and b allotypes (Fig. 2A). D23prod/+ mice maintained allelic exclusion, with all mature B lymphocytes expressing either IgMa or IgMb and no cells detectably expressing both IgH alleles. The presence of IgMb-expressing cells, albeit at low frequency (∼4%), was surprising, given that all pro-B cells in these mice initially carry a productive IgH knock-in of the a allotype. These cells inactivated the D23 IgH allele by VH replacement and underwent productive VHDHJH rearrangement of the WT IgH allele. FACS analysis cannot fully reveal the frequency of secondary rearrangement at the D23prod allele, however, because cells that undergo in-frame VH replacement remain IgMa+ and continue to express the IgM constant region from the targeted allele. In addition, cells that have undergone out-of-frame replacement may fail to productively rearrange the WT IgH locus and undergo apoptosis. Although the frequency of IgMb+ B cells in the secondary lymphoid tissues, such as spleen, mesenteric lymph nodes, and Peyer’s patches, was similar, with a slightly higher frequency observed in spleen (Fig. S1A), we consistently noted an increased frequency of IgMb+ cells in the marginal zone compared with follicular B cells (Fig. S1B). VH Replacement Leads to Inactivation of the D23prod Allele. To assess

the rearrangements occurring at the D23prod IgH allele, we isolated splenic B cells from D23prod heterozygous and homozygous mice. Using a mixture of degenerate forward primers specific for different families of VH genes (excluding the Q52 family, which includes the Q52.2.4 VH gene used in the D23prod rearrangement) with a reverse primer specific for the DH-JH junction of D23prod, we were able to amplify secondary rearrangements from the D23prod locus while avoiding amplification of the original D23prod allele or rearrangements from the WT IgH locus (Fig. 2B). Analysis of these sequences revealed that upstream VH genes had

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event that violates the 12/23 rule. VH replacement occurs between a 23-bp RSS of an upstream invading VH gene and a highly conserved 7-bp cryptic RSS (cRSS; TACTGTG) in the body of the recipient VH gene (6, 7). Because the cRSS is located near the 3′ terminus of the recipient VH gene, this process leaves behind only 5 bps proximal to the highly variable CDR3 region of the original VHDHJH joint, a minimal footprint that can be modified by exonuclease “chewback” and N-nucleotide addition (9, 10). VH replacement was initially observed in mouse B-cell lines in which a small fraction of cells regularly acquired alternate IgH rearrangements, thus “editing” their original specificity (6, 7). Since then, VH replacement has been studied in transgenic mouse models, human cell lines, and human blood. Recent studies that bioinformatically examined the frequency of 4- to 5-bp VH footprints at the VH-DH vs. DH-JH junctions have suggested evidence of VH replacement in at least 5% of the human Ig repertoire (9). Analysis of VH footprints in mouse IgH rearrangements revealed a similar frequency of VH replacement in wild-type (WT) mice, and also demonstrated a significantly higher frequency of VH replacement footprints in IgH sequences from patients with autoimmune diseases and animals of autoimmune-prone backgrounds (11, 12). However, the random nature of exonuclease and terminal deoxynucleotidyl transferase (TdT) activity at the CDR3 region of IgH joints makes it difficult to assess the contribution of VH replacement to the repertoire by sequence analysis, owing to loss of the VH footprint. Furthermore, our earlier work with a mouse model carrying a predefined nonproductive IgH rearrangement in the IgH locus has demonstrated that up to one-third of VH replacement events are mediated by sequence microhomology of the highly conserved 3′ bases of VH genes (4). Replacement reactions of this nature lack a detectable footprint and thus escape detection by sequence analysis. Although the nonproductive VHDHJH mouse model allowed us to study how VH replacement rescues nonproductive VHDHJH rearrangements, it did not address the contribution of VH replacement to the diversification of the primary immune repertoire or its role in editing self-reactive antibody specificities. Thus, we generated a knock-in mouse with a productive VHDHJH rearrangement knocked into its physiological position within the IgH locus. With this mouse model, we assessed the role of VH replacement in modifying productive IgH rearrangements in nonautoreactive and autoreactive settings.

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CD23 Fig. 1. Generation of the mouse model with a physiologically relevant productive IgH rearrangement. (A) Targeting scheme to generate an IgH locus that closely resembles a recombined VHDHJH allele. Targeting vector 1 was used to replace the JH elements with the D23prod VHDHJH joint and a single loxP site. Targeting vector 2 introduced a double loxP into the VH 81X site. Cre transduction followed by Southern blot analysis identified ES cells harboring both insertions on the same chromosome. Cre-mediated deletion resulted in the D23prod allele mimicking an endogenously rearranged IgH sequence. (B and C) Representative FACS analysis of bone marrow (B) and spleen (C). B-cell subsets of 7- to 8-wk-old WT and D23prod/+ mice demonstrate contraction of the pro–B-cell subset (c-kit+ CD25−) in D23prod/+. c-kit vs. CD25 plots are gated on B220+ IgM− bone marrow cells. AA4.1 vs. B220 plots are gated on B220+ CD19+ splenocytes. CD1d vs. CD23 plots are gated on B220+ AA4.1lo mature cells. Data are representative of more than five independent experiments, with more than five mice in each group.

invaded and replaced the recipient D23prod VH gene (Fig. 2C and Dataset S1). We observed in-frame as well as out-of-frame replacement events. Sequences also revealed extensive exonuclease E460 | www.pnas.org/cgi/doi/10.1073/pnas.1418001112

chewback of the replaced VH gene, occasionally eliminating the footprint of the original VH element, and N-nucleotide addition at the newly formed VH-to-VHDHJH joint. Sun et al.

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Invading VH VHD6.96 J558.42 7183.14

Sequence after VH Replacement OF ...TGTGCAAGGCTCCAGAGAGAACTCCGGCTAAGG... IF ...TGTGCAAGAGAGAACTCCGGCTAAGGTACTTTG... OF ...TGTGCAAGACAATCGAGAGAACTCCGGCTAAGG...

Fig. 2. Inactivation of the knock-in VHDHJH. (A) Splenic B cells examined by flow cytometry for expression of IgMa and IgMb allotype reflect the use of the knock-in and WT IgH allele, respectively. Cells were first gated on B220+ IgM+ expression. Data are representative of more than 10 independent experiments. (B) PCR strategy for amplifying VH replacement events from D23prod/+ mice. A combination of forward degenerate primers for VH genes, excluding the Q52 VH gene family and D23 VH gene, and a reverse primer specific for the D23 DH-JH sequence was used to selectively amplify VH replacement events at the knock-in locus. (C) Sequencing revealed both in-frame and out-of-frame VH replacement by the invading V gene (italicized nucleotides) with N-nucleotide addition (underlined nucleotides) and variable exonuclease chewback of the D23 VH footprint (bolded nucleotides).

We sorted IgMa+, IgMb+, and total B cells from D23prod/+ mice and sequenced the replacement joints of the D23prod allele. B cells expressing IgMb, and thus the WT Ig allele, carried exclusively out-of-frame VH replacements of the D23prod knock-in allele (Fig. 3); thus, the out-of-frame replacement events of the D23prod allele inactivated that allele, and the WT IgMb+ allele was subsequently rearranged and expressed. Replacement events amplified from IgMa+ B cells contained primarily in-frame replacements; the few out-of-frame replacement events were likely a result of contamination by IgMb+ cells. We previously described a mouse model in which nearly the entire Ig repertoire was generated as a result of VH replacement of the nonproductive D23stop allele. In that mouse model, we observed that ∼30% of the replacement events were mediated by sequence microhomology at the VH-to-VHDHJH junction and thus lacked N/P nucleotide addition (4). Sequences generated by replacement of the D23prod allele harbored N/P nucleotides in >95% of the cases, however. In the absence of any selection pressure, roughly two-thirds of such replacement events are expected to be out-of-frame, owing to the stochastic nature of exonuclease chewback and N-nucleotide addition. Our observation that the vast majority (∼95%) of replacement events from total B cells of D23prod/+ mice were out-of-frame suggests positive selection of cells expressing a rearrangement from the WT IgH allele, rather than a VH-replaced D23prod allele (Fig. 3). Preferential Use of Proximal V Genes in VH Replacement of the Productive VHDHJH Allele. Sequence analysis of invading VH genes

revealed a strong preference for genes in the VH7183 family, the upstream VH gene family most proximal to the D23prod rearrangement (Fig. 4). Indeed, the most frequently invading VH gene was the VH7183 family member VHD6.96, the VH gene immediately upstream of the knock-in site (Fig. S2 and Dataset S1). In contrast, in D23stop knock-in mice (4), the distribution of J558 and VH7183 donor VH genes in D23stop/D23stop replacement Sun et al.

events was similar to that of primary VDJ recombination at WT loci. One possible explanation for the preferential use of proximal VH genes in the replacement events at the D23prod locus was the limited window of opportunity for pro-B cells with a readily expressed VHDHJH to open the VH locus to initiate secondary recombination. Indeed, in D23prod/D23prod pro-B cells in which Igα signaling was abolished (ΔTm/ΔTm) (15), distal VH gene use was restored to levels observed in the primary WT IgH repertoire (Fig. 4B). Chromatin accessibility is a major mechanism regulating VHto-DHJH recombination (16). We suspected that VH gene use during VH replacement also might be regulated, at least in part, by chromatin accessibility. To evaluate this possibility, we quantified VH gene sterile transcripts extracted from pro-B cells of D23prod/ D23prod and D23stop/D23stop mice. These noncoding RNA transcripts of germline Ig genes reflect the accessibility of VH genes to RNA polymerase and RAG enzymes (17, 18). We compared J558 and VH7183 VH gene sterile transcripts by quantitative PCR using degenerate primers recognizing J558-specific and VH7183-specific genes but excluded the targeted VH gene (VH81x) and the D23prod knock-in VH gene (Q52.2.4). We observed a smaller fraction of J558 and more VH7183 sterile transcripts in D23prod/D23prod pro-B cells compared with D23stop/D23stop pro-B cells (Fig. S3). The correlation between differential sterile transcription and preferential proximal VH gene use in D23prod/D23prod B cells suggests a need for open chromatin and active sterile transcription to mediate VH replacement reactions. The Incidence of VH Replacement Is Not Increased During Receptor Editing. We next addressed the contribution of VH replacement

to editing of self-reactive BCR specificities. Receptor editing is central to rescuing B cells in which the IgH chain fails to pair with the IgL chain or the IgH and IgL chain pairing yields autoreactive specificities (19, 20). Previous studies have used transgenic mice with a VHDHJH joint knocked into the IgH locus PNAS | Published online January 21, 2015 | E461

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cifically recognizes the mouse Igκ constant region (23). As a result, developing B cells expressing Igκ antibody will bind to κ-macroself and undergo IgL chain editing. In D23prod/+ mice, Igλ+ B cells constitute

Fig. 6. Accumulation of B cells expressing the WT IgH in D23prod/+ mice with age. Splenic B cells from D23prod/+ mice of various ages were assessed for the proportion of B220+ cells expressing IgMb (WT Ig allele) by FACS analysis (n = 53). P < 0.0001, one-way ANOVA.

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thus expressed the WT IgH allele, underscoring the importance of VH replacement in diversification of the Ig repertoire in the knock-in mice and highlighting a possible selection mechanism for B cells as the mice age (Fig. S5).

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Fig. 5. Lack of increase in secondary IgH rearrangement in B cells from mice expressing ubiquitous anti-κ receptor. Editing of the D23prod allele was assessed in mice expressing a κ-macroself chimeric protein that cross-links Igκ. Splenic B cells from 9- to 10-wk-old D23prod and κ-macroself mice were assessed for surface expression of the IgL chain and IgM by flow cytometry. The increase in λ+ B cells highlights the dramatic editing at the IgL locus, whereas the lack of increase in WT IgH expression in D23prod/+ mice carrying the κ-macroself allele indicates no change in IgH receptor editing. Data are representative of three independent experiments, with at least four mice in each group.

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antibody repertoire in this mouse model. Furthermore, some B cells that maintained the expression of the knock-in allele altered the specificity of the D23prod rearrangement through productive replacement of the knock-in VH gene. The lack of B cells expressing IgH from both alleles indicated that D23prod/+ peripheral B cells maintain allelic exclusion. Sequencing of secondary VHDHJH rearrangements revealed evidence for 5′ exonuclease activity at the site of VH invasion and TdT activity that further diversified the VH-to-VHDHJH joint through N-nucleotide addition. Other groups have analyzed the sequences of mouse and human VHDHJH joints for footprints of VH replacement to assess the contribution of VH replacement to the antibody repertoire (9, 11, 12). These studies distinguished IgH genes with VH replacement by identifying 5- or 4-bp footprints at the VH-to-DH border and compared the frequency of VH footprints at VH-DH joints with the frequency at which these sequences are found at the DH-JH junction. However, because exonuclease and TdT activity in pro-B cells alter the VH-DH junction, such approaches likely underestimate the frequency of these events, as demonstrated by our finding of VH replacement sequences with footprints of ≤3 bp. In addition, our previous analysis of VHDHJH sequences from B cells of D23stop/D23stop mice demonstrated that many VH replacement events did not carry a footprint of the recipient VH gene, because VH replacement may occur via DNA microhomologymediated joining (4). The same study also highlighted that VH footprints are counterselected at the pre-B to immature transition owing to the presence of charged amino acids, specifically arginine, commonly encoded by VH replacement footprints, further suggesting that assessment of the VH replacement prevalence based on VH footprint detection may greatly underestimate its true frequency (4). Because the sequence of the recipient VHDHJH joint is defined in our D23prod knock-in mice, we can confidently identify all secondary rearrangement events and assess their contribution to this mouse model’s antibody repertoire. Our new mouse model allows us to directly assess the contribution of VH replacement to the further diversification of the primary B-cell repertoire, as well as its potential to edit autoimmune specificities. A significant fraction of VH replacement in D23stop/D23stop mice is mediated by microhomology between the invading and recipient VH genes, wherein the footprint of the original VH gene was missing and no N/P-nucleotides were observed (4). In contrast, only two out of more than 100 VH replacement events that we analyzed in D23prod mice appear to have been mediated by microhomology. The absence of VH replacement by

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microhomology in D23prod mice suggests that the replacement events occur exclusively in the presence of TdT. The high frequency of microhomology-mediated replacement events in D23stop/D23stop mice may either result from rearrangements occurring in the fetal liver in the absence of TdT or represent rare events in the bone marrow that are selected for preferentially owing to their lack of the VH footprint and thus the lower frequency of the positively charged arginine residues in CDR3 (4). The nearly complete absence of IgMb+ cells observed in newborn D23prod/+ mice suggests that any replacement events that do occur in the fetal liver of these mice are in-frame, consistent with the fact that TdT is not expressed during fetal hematopoiesis. However, because we failed to amplify any replacement joints from fetal livers of D23prod/+ mice, we conclude that replacement events at this stage of development are rare in this model. FACS analysis of B cells from adult D23prod/+ mice revealed that 3–5% of B cells were using the WT IgH allele, having inactivated the knock-in D23 allele by out-of-frame VH replacement. In addition, B cells that underwent an allele-inactivating VH replacement may fail to acquire an in-frame rearrangement on the WT IgH allele and thus perish in the bone marrow, so that the frequency of replacement events may be even higher. We could not assess the true extent of VH replacement until we also examined the frequency of in-frame VH replacement, however. In D23prod/+ total splenic B cells, we observed that out-of-frame replacement events were far more frequent than in-frame secondary rearrangements, with inframe VH replacement accounting for only 5% of secondary rearrangements. Owing to the stochastic nature of TdT and exonuclease activity, only two-thirds of VH replacement events are expected to be out-of-frame, but since the vast majority were out-of-frame, there must be selection against in-frame and/or selection for out-of-frame replacement. In-frame VH replacements may be selected against due to the prevalence of charged amino acids encoded by the footprint bases (as discussed above). On the other hand, the out-of-frame replacement events allow de novo rearrangement of the WT IgH locus, and thus significantly diversify the antibody repertoire, from which specificities expressed in peripheral B cells can be selected. Indeed, we observed steady accumulation of B cells that had undergone out-of-frame VH replacement and now expressed IgMb (WT) heavy chain as the animals aged; it was this population that was preferentially recruited into germinal centers of gut-associated immune organs. The preferential participation of IgMb+ B cells in the gut-associated germinal centers likely reflects the broader antibody repertoire of this population. The accumulation of such “escapee” cells with age is frequently observed in IgH knock-in mouse models. Most VH replacements at the D23prod allele used VH genes from the VH7183 V gene family, predominantly VHD6.96, the VH gene most proximal to the knock-in site. This finding is in striking contrast to our previous study of D23stop/D23stop mice, where the entire Ig repertoire was generated by secondary rearrangements of the nonproductive IgH alleles. In that case, VH replacement events used the full spectrum of VH genes, and the repertoire of VH elements used closely resembled that seen in primary WT IgH rearrangements (4). Pro-B cells blocked from progressing in development, such as those from D23stop/D23stop mice or from Igα signaling-deficient animals, have a broader window of opportunity to undergo secondary rearrangement. In contrast, cells that undergo VH replacement in D23prod animals likely initiate secondary IgH rearrangement before receiving a signal through the pre-BCR. The highly restricted VH use during replacement observed in the D23prod mice may reflect a lack of contraction at the IgH locus during the limited time that these cells remain in the E464 | www.pnas.org/cgi/doi/10.1073/pnas.1418001112

pro–B-cell compartment and may be related to chromatin inaccessibility of the distal VH genes in the early stages of B-cell differentiation (25, 26). This hypothesis is supported by the observed disparity in sterile transcription of J558 and VH7183 gene segments in pro-B cells from D23stop/D23stop mice vs. D23prod/D23prod mice. The strong bias toward proximal VH gene use disappears in D23prod mice with a defect in Igα signaling, likely because the VH locus becomes more accessible as a consequence of the blocked progression from the pro–B- to the pre–B-cell stage. The observed bias toward DH-proximal VH genes in D23prod mice is consistent with the fact that other mouse models harboring productive IgH transgenes show inefficient exclusion of the most 3′ VH to DH gene, suggesting that the 3′ end of the IgH locus is accessible during early pro–B-cell development (27–29). Furthermore, because theVH-DH region harbors several CTCFbinding elements critical for allelic exclusion and for regulation of IgH locus accessibility (30), it is conceivable that the loss of CTCF sites or other locus regulatory elements resulting from our gene targeting strategy increases the accessibility of the 3′ VH genes prematurely, thus allowing VH replacement to occur earlier then it would in WT pro-B cells. Along with diversifying the antibody repertoire, VH replacement has been suggested as a mechanism of receptor editing in immature B cells (11, 22, 31). We examined the frequency of VH replacement of the D23prod allele on two autoreactive backgrounds, first by pairing this IgH with its cognate IgL chain D23κ, resulting in an antibody with ssDNA/dsDNA autospecificity, and then by expressing the D23prod knock-in in mice that ubiquitously express a protein that cross-links all BCRs that carry a Igκ, thus triggering massive IgL editing. In both cases, we observed no change in the frequency of VH replacement. Although a recent study found evidence of VH replacement breaks occurring in B cells that recently emigrated from the bone marrow (22), the presence of N-nucleotides in ∼99% of VH replacement joints analyzed from D23prod mice suggests that replacement occurs at the pro–B-cell stage, at which the expression of Tdt is limited (32). Our conclusion is consistent with an earlier analysis of cRSS cleavage throughout the B-cell developmental stages (33). Our finding that the presence of a prerearranged D23κ did not alter the frequency of VH replacement on the D23prod IgH chain further suggests that this process occurs in early B-cell progenitors, before IgL chain expression. The increased frequency of VH replacement footprints in autoimmune repertoires may reflect positive selection of the additional positively charged amino acids encoded by the VH footprint in the CDR3 region (4, 34), rather than evidence suggesting VH replacement as a mechanism for editing of the primary repertoire in response to autoantigens. The majority of earlier IgH knock-in mice were of limited use in the study of secondary IgH rearrangements, because these mice had VHDHJH joints knocked-in ∼180 kb downstream of the most 3′ VH gene, replacing the endogenous JH elements and leaving the intervening DH elements intact. In contrast, the D23prod allele closely resembled an endogenous IgH rearrangement, allowing us to study the secondary rearrangement of a productive VHDHJH joint under quasi-physiological conditions. The use of mice generated by nuclear transfer using B cells as donors, as well as mice produced using iPS cells following the reprogramming of mature B lymphocytes, may provide further insight into VH replacement, because B-cell progenitors in these mice will have prerearranged Ig loci (35–37). Indeed, studies by the Casola group (see the accompanying paper by Kumar et al., ref. 38) reveal that animals generated by transnuclear transfer from an IgA+ plasma cells have peripheral B cells with a very high frequency of VH replacement of the original IgH rearrangement. Those studies demonstrated that the contribution of VH replacement to the diversification of the primary immune repertoire is not limited to the D23prod knock-in mice. The Sun et al.

Materials and Methods Gene Targeting. Our gene targeting strategy was similar to the previously published D23stop targeting strategy (4). The rearranged D23 IgH gene segment was isolated by PCR amplification, and its promoter was cloned by a PCR walking strategy. The D23 VDJ together with the promoter was cloned into the targeting vector 1 (Fig. 1A) containing a floxed neo cassette flanked by homology to the JH region. To generate targeting vector 2 (Fig. 1A), a double LoxP followed by an frt-flanked neomycin-resistance cassette was inserted into a vector containing homology to the VH 81X gene. (The orientation of the VH element was not known at the time of targeting.) Sequential targeting of first the double LoxP and then the D23 VDJ resulted in the targeted IgH locus. The chimera with transmission of the targeted allele into the germline was crossed to the deleter strain to eliminate the intervening DH elements by Cre activity in germ cells (39). Preparative and Analytical FACS. Fluorescence staining was performed as described previously (4). Cells isolated from κ-macroself mice were incubated in 1. Tonegawa S (1983) Somatic generation of antibody diversity. Nature 302(5909):575–581. 2. Melchers F, et al. (1994) Roles of IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage. Annu Rev Immunol 12(1):209–225. 3. Herzog S, Reth M, Jumaa H (2009) Regulation of B cell proliferation and differentiation by pre-B-cell receptor signalling. Nat Rev Immunol 9(3):195–205. 4. Koralov SB, Novobrantseva TI, Königsmann J, Ehlich A, Rajewsky K (2006) Antibody repertoires generated by VH replacement and direct VH to JH joining. Immunity 25(1):43–53. 5. Lutz J, Müller W, Jäck HM (2006) VH replacement rescues progenitor B cells with two nonproductive VDJ alleles. J Immunol 177(10):7007–7014. 6. Kleinfield R, et al. (1986) Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma. Nature 322(6082):843–846. 7. Reth M, Gehrmann P, Petrac E, Wiese P (1986) A novel VH to VHDJH joining mechanism in heavy-chain-negative (null) pre-B cells results in heavy-chain production. Nature 322(6082):840–842. 8. Jung D, Giallourakis C, Mostoslavsky R, Alt FW (2006) Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu Rev Immunol 24:541–570. 9. Zhang Z, et al. (2003) Contribution of Vh gene replacement to the primary B-cell repertoire. Immunity 19(1):21–31. 10. Zhang Z, Burrows PD, Cooper MD (2004) The molecular basis and biological significance of VH replacement. Immunol Rev 197(1):231–242. 11. Huang L, et al. (2013) Contribution of V(H) replacement products in mouse antibody repertoire. PLoS ONE 8(2):e57877. 12. Lange MDH, et al. (2014) Accumulation of VH replacement products in IgH genes derived from autoimmune diseases and anti-viral responses in human. Front Immunol 5:345. 13. Baccala R, Quang TV, Gilbert M, Ternynck T, Avrameas S (1989) Two murine natural polyreactive autoantibodies are encoded by nonmutated germ-line genes. Proc Natl Acad Sci USA 86(12):4624–4628. 14. Koralov SB, Novobrantseva TI, Hochedlinger K, Jaenisch R, Rajewsky K (2005) Direct in vivo VH to JH rearrangement violating the 12/23 rule. J Exp Med 201(3):341–348. 15. Torres RM, Flaswinkel H, Reth M, Rajewsky K (1996) Aberrant B-cell development and immune response in mice with a compromised BCR complex. Science 272(5269): 1804–1808. 16. Roth DB, Roth SY (2000) Unequal access: Regulating V(D)J recombination through chromatin remodeling. Cell 103(5):699–702. 17. Alessandrini A, Desiderio SV (1991) Coordination of immunoglobulin DJH transcription and D-to-JH rearrangement by promoter-enhancer approximation. Mol Cell Biol 11(4): 2096–2107.

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PNAS PLUS

unlabeled mouse IgG/κ antibody for blocking before antibody staining. Cell sorting was performed using a triple-laser flow cytometer (BD FACSVantage) and a four-laser flow cytometer (BD FACSAria). Analytical FACS was carried out on a four-laser flow cytometer (BD LSRFortessa and LSRII). PCR and RT-PCR. DNA from sorted populations was prepared by proteinase K digestion in Tris buffer for 3 h. IgH rearrangements were amplified using a mix of previously described forward primers and a reverse primer specific to the D23H D–J sequence (5′-CCCCAGTAGTCAAAGTACC-3′) or a JH2 primer (5′AGACTGTGAGAGTGGTGCC-3′) (40). High-fidelity polymerase was used to minimize point mutations during the PCR analyses (Roche). The expectedsized band (∼380 bp) was excised and gel-purified before being inserted into the pGEM-T easy vector by TA cloning (Promega). The subcloned DNA was prepared by column purification (Qiagen) and sequenced using T7 primer (Macrogen). RNA was isolated from sorted populations by column purification (Qiagen RNeasy Micro Kit). cDNA was prepared immediately using random hexamer primers (Superscript VILO; Life Technologies). Forward and reverse primers specific to but degenerate within the J558 (forward: 5′-CTTCTGGCTACACCTTCAC-3′; reverse: 5′-CTGAGCTGCATGTAGGCTG-3′) and 7183 (forward: 5′- GCCTCTGGATTCACTTTCAG-3′; reverse: 5′-CATTGTCTCTGGAGATGGTG-3′) V gene families were used to quantitatively amplify germline transcription of V genes. Mice. Mice were housed and cared for under specific pathogen-free conditions in accordance with protocols approved by the Animal Care and Use Committees of Harvard Medical School and NYU School of Medicine. Sequence Analysis of IgH Rearrangements. VDJ sequences were analyzed using the National Center for Biotechnology Information’s IgBlast program (Dataset S1). ACKNOWLEDGMENTS. We thank Stefano Casola for the open and enjoyable discussions on the complementary projects. This work was supported by the Arnold and Mabel Beckman Foundation, the Ralph S. French Charitable Foundation, and the National Institutes of Health [Grant R21AI110830 (to S.B.K.)]. A.S. was supported by National Institutes of Health Grant 5T32GM007308-36 and National Cancer Institute Grant 5T32CA009161-39.

18. Yancopoulos GD, Blackwell TK, Suh H, Hood L, Alt FW (1986) Introduced T- cell receptor variable region gene segments recombine in pre-B cells: Evidence that B and T cells use a common recombinase. Cell 44(2):251–259. 19. Nemazee D (1999) Receptor editing in B cells. Advances in Immunology, ed Frank JD (Academic Press, San Diego), Vol 74, pp 89–126. 20. Halverson R, Torres RM, Pelanda R (2004) Receptor editing is the main mechanism of B-cell tolerance toward membrane antigens. Nat Immunol 5(6):645–650. 21. Chen C, Nagy Z, Prak EL, Weigert M (1995) Immunoglobulin heavy chain gene replacement: A mechanism of receptor editing. Immunity 3(6):747–755. 22. Liu J, et al. (2013) Regulation of VH replacement by B-cell receptor-mediated signaling in human immature B cells. J Immunol 190(11):5559–5566. 23. Ait-Azzouzene D, et al. (2005) An immunoglobulin C κ-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system. J Exp Med 201(5):817–828. 24. Dildrop R, Gause A, Müller W, Rajewsky K (1987) A new V gene expressed in lambda-2 light chains of the mouse. Eur J Immunol 17(5):731–734. 25. Fuxa M, et al. (2004) Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev 18(4):411–422. 26. Stanhope-Baker P, Hudson KM, Shaffer AL, Constantinescu A, Schlissel MS (1996) Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell 85(6):887–897. 27. Kurosawa Y, et al. (1981) Identification of D segments of immunoglobulin heavychain genes and their rearrangement in T lymphocytes. Nature 290(5807):565–570. 28. Iacomini J, Yannoutsos N, Bandyopadhay S, Imanishi-Kari T (1991) Endogenous immunoglobulin expression in mu transgenic mice. Int Immunol 3(2):185–196. 29. Costa TE, Suh H, Nussenzweig MC (1992) Chromosomal position of rearranging gene segments influences allelic exclusion in transgenic mice. Proc Natl Acad Sci USA 89(6): 2205–2208. 30. Guo C, et al. (2011) CTCF-binding elements mediate control of V(D)J recombination. Nature 477(7365):424–430. 31. Itoh K, et al. (2000) Immunoglobulin heavy chain variable region gene replacement as a mechanism for receptor revision in rheumatoid arthritis synovial tissue B lymphocytes. J Exp Med 192(8):1151–1164. 32. Li YS, Hayakawa K, Hardy RR (1993) The regulated expression of B lineage- associated genes during B-cell differentiation in bone marrow and fetal liver. J Exp Med 178(3): 951–960.

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frequency of replacement events may vary between the two models owing to the differences in VHDHJH substrate sequence, the chromosomal location of the primary rearrangement relative to the available donor VH genes or in relation to transcription regulatory sites, or to differential signaling properties of IgM and IgA cytoplasmic tails. In summary, our analysis of secondary rearrangements in a mouse model with a naturally positioned IgH knock-in demonstrates that VH replacement can modify productively rearranged VHDHJH joints as a mechanism of antibody repertoire diversification. Extrapolating from the D23prod allele to the entire antibody repertoire, the overall contribution of this secondary diversification process is apparently minor, and the physiological impact of VH replacement may be more significant for rescuing B-cell progenitors with nonproductive IgH rearrangements (4). In either case, however, VH replacement occurs in B progenitor cells independent of antigen specificity and is unlikely to contribute to receptor editing in the context of the counterselection of autoreactive BCRs in pre-B or immature B lymphocytes.

33. Davila M, et al. (2007) Multiple, conserved cryptic recombination signals in VH gene segments: Detection of cleavage products only in pro-B cells. J Exp Med 204(13): 3195–3208. 34. Sekiguchi DR, Eisenberg RA, Weigert M (2003) Secondary heavy chain rearrangement: A mechanism for generating anti-double-stranded DNA B cells. J Exp Med 197(1):27–39. 35. Dougan SK, et al. (2013) Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus. Nature 503(7476):406–409. 36. Dougan SK, et al. (2012) IgG1+ ovalbumin-specific B-cell transnuclear mice show class switch recombination in rare allelically included B cells. Proc Natl Acad Sci USA 109(34):13739–13744.

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37. Hanna J, et al. (2008) Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133(2):250–264. 38. Kumara R, et al. (2015) Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell. Proc Natl Acad Sci USA 112: E450–E457. 39. Schwenk F, Baron U, Rajewsky K (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res 23(24):5080–5081. 40. Ehlich A, Martin V, Müller W, Rajewsky K (1994) Analysis of the B-cell progenitor compartment at the level of single cells. Curr Biol 4(7):573–583.

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