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In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease 1 . RAG orchestrates Igh V(D)J recombination upon capturing a J H -RSS within the J H -RSS-based recombination centre 1-3 (RC). J H -RSS orientation programmes RAG to scan upstream D- and V H -containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion 4-7 . During Igh scanning, RAG robustly utilizes only D-RSSs or V H -RSSs in convergent (deletional) orientation with J H -RSSs 4-7 . However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters 8 , inconsistent with linear scanning 2 . Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements 9,10 , which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element 11 . Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element 12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to V H  locus inversion 7 , DJ H inversion abrogated V H -to-DJ H joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-J H and D-to-Jκ rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating V H joining by RAG-scanning impediments., (© 2024. The Author(s).)

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin (Ig) heavy chain (IgH) and light chain (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation. Based on these principles, we developed a humanized mouse model approach to diversify an anti-PD1 therapeutic antibody and allow isolation of variants with novel properties. In this approach, component Ig gene segments of the anti-PD1 antibody underwent de novo V(D)J recombination to diversify the anti-PD1 antibody in the primary antibody repertoire in the mouse models. Immunization of these mouse models further modified the anti-PD1 antibodies through SHM. Known anti-PD1 antibodies block interaction of PD1 with its ligands to alleviate PD1-mediated T cell suppression, thereby boosting antitumor T cell responses. By diversifying one such anti-PD1 antibody, we derived many anti-PD1 antibodies, including anti-PD1 antibodies with the opposite activity of enhancing PD1/ligand interaction. Such antibodies theoretically might suppress deleterious T cell activities in autoimmune diseases. The approach we describe should be generally applicable for diversifying other therapeutic antibodies., Competing Interests: Competing interest statement: M.T., H.-L.C., and F.W.A. have filed a patent application on the new anti-PD1 antibodies described in the manuscript, and F.W.A. is a cofounder of Otoro Biopharmaceuticals.

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a J H -recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs 1,2 . The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning 3 . Scanning of RAG from the DJ H -RC-RSS to upstream convergent V H -RSSs is impeded by D-proximal CTCF-binding elements (CBEs) 2-5 . Primary progenitor B cells undergo a mechanistically undefined contraction of the V H locus that is proposed to provide distal V H s access to the DJ H -RC 6-9 . Here we report that an inversion of the entire 2.4-Mb V H locus in mouse primary progenitor B cells abrogates rearrangement of both V H -RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic V H -RSSs that are normally in opposite orientation and RAG scanning beyond the V H locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the V H locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL) 10 , a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the V H locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.

IgH class switch recombination (CSR) replaces Cμ constant region (C H ) exons with one of six downstream C H s by joining transcription-targeted double-strand breaks (DSBs) in the Cμ switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3'IgH regulatory region (3'IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are 10 consecutive CTCF-binding elements (CBEs) downstream of the 3'IgHRR, termed the "3'IgH CBEs." Prior studies showed that deletion of eight 3'IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3'IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except that of Sγ1. Moreover, deletion of all 3'IgH CBEs rendered the 6-kb region just downstream highly transcribed and caused sequences within to be aligned with Sμ, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3'IgH CBEs as critical insulators for focusing loop extrusion-mediated 3'IgHRR transcriptional and CSR activities on upstream C H locus targets., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As a master regulator of functional Ig heavy chain (IgH) expression, the IgH 3' regulatory region (3'RR) controls multiple transcription events at various stages of B-cell ontogeny, from newly formed B cells until the ultimate plasma cell stage. The IgH 3'RR plays a pivotal role in early B-cell receptor expression, germ-line transcription preceding class switch recombination, interactions between targeted switch (S) regions, variable region transcription before somatic hypermutation, and antibody heavy chain production, but the functional ranking of its different elements is still inaccurate, especially that of its evolutionarily conserved quasi-palindromic structure. By comparing relevant previous knockout (KO) mouse models (3'RR KO and hs3b-4 KO) to a novel mutant devoid of the 3'RR quasi-palindromic region (3'PAL KO), we pinpointed common features and differences that specify two distinct regulatory entities acting sequentially during B-cell ontogeny. Independently of exogenous antigens, the 3'RR distal part, including hs4, fine-tuned B-cell receptor expression in newly formed and naïve B-cell subsets. At mature stages, the 3'RR portion including the quasi-palindrome dictated antigen-dependent locus remodeling (global somatic hypermutation and class switch recombination to major isotypes) in activated B cells and antibody production in plasma cells.

During B-cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200 kilobases downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis Igh organizational features in combination with frequent S-region DSBs initiated by AID. We further implicate ATM-dependent DSB-response factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.

Ig heavy chain (IgH) variable region exons are assembled from V, D, and J gene segments during early B-lymphocyte differentiation. A several megabase region at the "distal" end of the mouse IgH locus (Igh) contains hundreds of V(H)s, separated by an intergenic region from Igh Ds, J(H)s, and constant region exons. Diverse primary Igh repertoires are generated by joining Vs, Ds, and Js in different combinations, with a given B cell productively assembling only one combination. The intergenic control region 1 (IGCR1) in the V(H)-to-D intergenic region regulates Igh V(D)J recombination in the contexts of developmental order, lineage specificity, and feedback from productive rearrangements. IGCR1 also diversifies IgH repertoires by balancing proximal and distal V(H) use. IGCR1 functions in all these regulatory contexts by suppressing predominant rearrangement of D-proximal V(H)s. Such IGCR1 functions were neutralized by simultaneous mutation of two CCCTC-binding factor (CTCF)-binding elements (CBE1 and CBE2) within it. However, it was unknown whether only one CBE mediates IGCR1 functions or whether both function in this context. To address these questions, we generated mice in which either IGCR1 CBE1 or CBE2 was replaced with scrambled sequences that do not bind CTCF. We found that inactivation of CBE1 or CBE2 individually led to only partial impairment of various IGCR1 functions relative to the far greater effects of inactivating both binding elements simultaneously, demonstrating that they function cooperatively to achieve full IGCR1 regulatory activity. Based on these and other findings, we propose an orientation-specific looping model for synergistic CBE1 and CBE2 functions.

Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100-200 kb apart within switch (S) regions in the immunoglobulin heavy-chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sμ and in a downstream acceptor S region, with a DSB in Sμ being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B-cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sγ1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sγ1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sμ and Sγ1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intrachromosomal DSBs separated by several hundred kilobases to be frequently joined together and discuss the relevance of this finding for recurrent interstitial deletions in cancer.

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D(H)) and joining (J(H)) gene segments were methylated, DJ(H) junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eμ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination., Competing Interests: The authors have declared that no competing interests exist.

Genes encoding immunoglobulin heavy chains (Igh) are assembled by rearrangement of variable (V(H)), diversity (D(H)) and joining (J(H)) gene segments. Three critical constraints govern V(H) recombination. These include timing (V(H) recombination follows D(H) recombination), precision (V(H) gene segments recombine only to DJ(H) junctions) and allele specificity (V(H) recombination is restricted to DJ(H)-recombined alleles). Here we provide a model for these universal features of V(H) recombination. Analyses of DJ(H)-recombined alleles showed that DJ(H) junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG recombinase proteins, which thereby permitted D(H)-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that V(H) recombination is precise, because these changes did not extend to germline D(H) segments located 5' of the DJ(H) junction.

Induced pluripotent stem cells (iPSCs) can be formed from somatic cells by a defined set of genetic factors; however, aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster often hinders their developmental potency and ability to contribute to high-grade chimerism in mice. Here, we describe an approach that allows splenic B cells activated to undergo Ig heavy-chain (IgH) class-switch recombination (CSR) to be reprogrammed into iPSCs that contribute to high-grade chimerism in mice. Treatment of naïve splenic B cells in culture with anti-CD40 plus IL-4 induces IgH CSR from IgM to IgG1 and IgE. CSR leads to irreversible IgH locus deletions wherein the IgM-producing Cμ exons are permanently excised from the B-cell genome. We find that anti-CD40 plus IL-4-activated B cells produce iPSCs that are uniformly hypermethylated in the imprinted Dlk1-Dio3 gene cluster and fail to produce chimerism in mice. However, treatment of activated B cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine before and at early stages of reprogramming attenuates hypermethylation of the Dlk1-Dio3 locus in resultant iPSCs and enables them to form high-grade chimerism in mice. These conditions allowed us to produce chimeric mice in which all mature B cells were derived entirely from IgG1-expressing B-cell-derived iPSCs. We conclude that culture conditions of activated B cells before and at early stages of reprogramming influence the developmental potency of resultant iPSCs.

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) heavy-chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and nontemplate strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA-processing/degradation complex, in targeting AID to both DNA strands. In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ig and T-cell receptor (TCR) variable-region gene exons are assembled from component variable (V), diversity (D) and joining (J) gene segments during early B and T cell development. The RAG1/2 endonuclease initiates V(D)J recombination by introducing DNA double-strand breaks at borders of the germ-line segments. In mice, the Ig heavy-chain (IgH) locus contains, from 5' to 3', several hundred V(H) gene segments, 13 D segments, and 4 J(H) segments within a several megabase region. In developing B cells, IgH variable-region exon assembly is ordered with D to J(H) rearrangement occurring on both alleles before appendage of a V(H) segment. Also, IgH V(H) to DJ(H) rearrangement does not occur in T cells, even though DJ(H) rearrangements occur at low levels. In these contexts, V(D)J recombination is controlled by modulating substrate gene segment accessibility to RAG1/2 activity. To elucidate control elements, we deleted the 100-kb intergenic region that separates the V(H) and D clusters (generating ΔV(H)-D alleles). In both B and T cells, ΔV(H)-D alleles initiated high-level antisense and, at lower levels, sense transcription from within the downstream D cluster, with antisense transcripts extending into proximal V(H) segments. In developing T lymphocytes, activated germ-line antisense transcription was accompanied by markedly increased IgH D-to-J(H) rearrangement and substantial V(H) to DJ(H) rearrangement of proximal IgH V(H) segments. Thus, the V(H)-D intergenic region, and likely elements within it, can influence silencing of sense and antisense germ-line transcription from the IgH D cluster and thereby influence targeting of V(D)J recombination.

The 5' end of the IgH locus contains a cluster of DNaseI hypersensitive sites, one of which (HS1) was shown to be pro-B cell specific and to contain binding sites for the transcription factors PU.1, E2A, and Pax5. These data as well as the location of the hypersensitive sites at the 5' border of the IgH locus suggested a possible regulatory function for these elements with respect to the IgH locus. To test this notion, we generated mice carrying targeted deletions of either the pro-B cell specific site HS1 or the whole cluster of DNaseI hypersensitive sites. Lymphocytes carrying these deletions appear to undergo normal development, and mutant B cells do not exhibit any obvious defects in V(D)J recombination, allelic exclusion, or class switch recombination. We conclude that deletion of these DNaseI hypersensitive sites does not have an obvious impact on the IgH locus or B cell development.

Ig heavy chain (IgH) class-switch recombination (CSR) replaces the IgH C mu constant region exons with one of several sets of downstream IgH constant region exons (e.g., C gamma, C epsilon, or C alpha), which affects switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). Activation-induced cytidine deaminase (AID) initiates CSR by promoting DNA double-strand breaks (DSBs) within switch (S) regions flanking the donor C mu (S mu) and a downstream acceptor C(H) (e.g., S gamma, S epsilon, S alpha) that are then joined to complete CSR. DSBs generated in S mu frequently are joined within S mu to form internal switch region deletions (ISD). AID-induced ISD and mutations have been considered rare in downstream S regions, suggesting that AID targeting to these S regions requires its prior recruitment to S mu. We have now assayed for CSR and ISD in B cells lacking S mu (S mu(-/-) B cells). In S mu(-/-) B cells activated for CSR to IgG1 and IgE, CSR to IgG1 was greatly reduced; but, surprisingly, CSR to IgE occurred at nearly normal levels. Moreover, normal B cells had substantial S gamma1 ISD and increased mutations in and near S gamma1, and levels of both were greatly increased in S mu(-/-) B cells. Finally, S mu(-/-) B cells underwent downstream CSR between S gamma1 and S epsilon on alleles that lacked S mu CSR to these sequences. Our findings show that AID targets downstream S regions independently of CSR with Smu and implicate an alternative pathway for IgE class switching that involves generation and joining of DSBs within two different downstream S regions before S mu joining.

A tissue-specific transcriptional enhancer, Emu, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Emu results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Emu-independent and Emu-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cmu exons with a set of downstream IgH constant region (C(H)) exons. Individual sets of C(H) exons are flanked upstream by long (1-10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Smu region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region-specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sgamma1 region was replaced with wild-type (WT) or synthetic 2-kb Sgamma3 sequences or a synthetic 2-kb Sgamma1 sequence. We found that both the inserted endogenous and synthetic Sgamma3 sequences functioned similarly to a size-matched synthetic Sgamma1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sgamma3 can function similarly to Sgamma1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype-specific functions of other endogenous S regions.

Activation-induced cytosine deaminase (AID) is essential for both somatic hypermutation (SHM) and class switch recombination (CSR), two processes involved in antibody diversification. Previously, various groups showed both in vitro and in vivo that AID initiates SHM and CSR by deaminating cytosines in DNA in a transcription-dependent manner. Although in vivo both DNA strands are equally targeted by AID, many in vitro and bacterial experiments found that AID almost exclusively targets the nontemplate strand of a transcribed substrate. Here, we report the detection of antisense transcripts in assembled Ig heavy chain (IgH) variable region exons and their immediate downstream region, as well as in switch regions, sequences that, respectively, are targets for SHM and CSR in vivo. In contrast, we did not detect antisense transcripts from the Cmu constant region exons, which lie between the IgH variable region exons and downstream S regions and which are not normally an AID target. Expression of the antisense variable region/flanking region and the S-region transcripts were found in all lymphocytes that transcribe these sequences in the sense direction. Steady-state levels of antisense transcripts appeared very low, and start sites potentially appeared heterogeneous. We discuss the potential implications of antisense IgH locus transcription for AID targeting or other processes.

To mount an optimum immune response, mature B lymphocytes can change the class of expressed antibody from IgM to IgG, IgA, or IgE through a recombination/deletion process termed immunoglobulin heavy chain (IgH) class switch recombination (CSR). CSR requires the activation-induced cytidine deaminase (AID), which has been shown to employ single-stranded DNA as a substrate in vitro. IgH CSR occurs within and requires large, repetitive sequences, termed S regions, which are parts of germ line transcription units (termed "C(H) genes") that are composed of promoters, S regions, and individual IgH constant region exons. CSR requires and is directed by germ line transcription of participating C(H) genes prior to CSR. AID deamination of cytidines in S regions appears to lead to S region double-stranded breaks (DSBs) required to initiate CSR. Joining of two broken S regions to complete CSR exploits the activities of general DNA DSB repair mechanisms. In this chapter, we discuss our current knowledge of the function of S regions, germ line transcription, AID, and DNA repair in CSR. We present a model for CSR in which transcription through S regions provides DNA substrates on which AID can generate DSB-inducing lesions. We also discuss how phosphorylation of AID may mediate interactions with cofactors that facilitate access to transcribed S regions during CSR and transcribed variable regions during the related process of somatic hypermutation (SHM). Finally, in the context of this CSR model, we further discuss current findings that suggest synapsis and joining of S region DSBs during CSR have evolved to exploit general mechanisms that function to join widely separated chromosomal DSBs.

V(D)J recombination assembles antigen receptor variable region genes from component germline variable (V), diversity (D), and joining (J) gene segments. For B cells, such rearrangements lead to the production of immunoglobulin (Ig) proteins composed of heavy and light chains. V(D)J is tightly controlled at the Ig heavy chain locus (IgH) at several different levels, including cell-type specificity, intra- and interlocus ordering, and allelic exclusion. Such controls are mediated at the level of gene segment accessibility to V(D)J recombinase activity. Although much has been learned, many long-standing questions regarding the regulation of IgH locus rearrangements remain to be elucidated. In this review, we summarize advances that have been made in understanding how V(D)J recombination at the IgH locus is controlled and discuss important areas for future investigation.

Studies of chimeric mice demonstrated that the core Ig heavy chain (IgH) intronic enhancer (iEmu) functions in V(D)J and class switch recombination at the IgH locus. To more fully evaluate the role of this element in these and other processes, we generated mice homozygous for germ-line mutations in which the core sequences of iEmu (cEmu) were either deleted (cEmu(Delta/Delta) mice) or replaced with a pgk-Neo(R) cassette (cEmu(N/N) mice). The cEmu(Delta/Delta) mice had reduced B cell numbers, in association with impaired D to J(H) and V(H) to DJ(H) rearrangement, whereas cEmu(N/N) mice had a complete block in IgH V(D)J(H) recombination, confirming that additional cis elements cooperate with iEmu to enforce D to J(H) recombination. In addition, developing cEmu(Delta/Delta) and cEmu(N/N) B lineage cells had correspondingly decreased levels of germ-line transcripts from the J(H) region of the IgH locus (mu0 and Imu transcripts); although both had normal levels of germ-line V(H) transcripts, suggesting that cEmu may influence IgH locus V(D)J recombination by influencing accessibility of J(H) proximal regions of the locus. Consistent with chimera studies, peripheral cEmu(Delta/Delta) B cells had normal surface Ig and relatively normal class switch recombination. However, cEmu(Delta/Delta) B cells also had relatively normal somatic hypermutation of their IgH variable region genes, showing unexpectedly that the cEmu is not required for this process. The availability of mice with the iEmu mutation in their germ line will facilitate future studies to elucidate the roles of iEmu in V(H)(D)J(H) recombination in the context of IgH chromatin structure and germ-line transcription.

Ig heavy chain class switch recombination (CSR) involves a recombination/deletion mechanism that exchanges the expressed C(H) gene with a downstream C(H) gene. CSR is mediated by highly repetitive switch (S) region sequences and requires the activation-induced deaminase (AID). The S region 5' of the C mu gene (S mu) can undergo high-frequency internal deletions in normal B cells and B cell lines activated for CSR, although the relationship of these deletions and CSR has not been elucidated. In this study, we introduced constitutively transcribed S mu or S gamma 2b regions into a pro-B cell line that can be activated for AID expression, CSR, and endogenous S mu deletions. We find that randomly integrated S region transcription units in these cells also undergo increased levels of internal rearrangements after cellular activation, indicating that the deletion process is independent of location within the Ig heavy chain locus and potentially AID-promoted. To test the latter issue, we generated hybridomas from wild-type and AID-deficient activated B cells and assayed them for internal S mu deletions and S region mutations. These studies demonstrated that efficient intra-S region recombination depends on AID expression and that internal S region deletions are accompanied by frequent mutations, indicating that most S region deletions occur by the same mechanism as CSR.

To assess the role of the DNA-PKcs nonhomologous DNA end-joining (NHEJ) protein in Ig heavy chain class switch recombination (CSR), we assayed CSR ability of DNA-PKcs-deficient (DP-T) B cells generated via complementation of DP-T mice with Ig heavy chain and light chain knock-in transgenes (DP-T/HC/LC mice). DP-T/HC/LC mice were severely deficient for all serum IgH isotypes except IgM and, unexpectedly, IgG1. Upon appropriate stimulation, DP-T/HC/LC B cells showed normal proliferation, germline C(H) gene transcription, and AID induction, indicating that DNA-PKcs deficiency did not affect cellular events upstream to CSR. Yet, in vitro activated DP-T/HC/LC B cells again underwent switching only to IgG1 and, like wild-type cells, frequently underwent CSR to gamma1 on both chromosomes. We conclude that DNA-PKcs is required for CSR to most C(H) genes but that CSR to gamma1 occurs via a DNA-PKcs-independent mechanism.

The Ig heavy chain (IgH) constant region (CH) genes are organized from 5' to 3' in the order Cmicro, Cdelta, Cgamma3, Cgamma1, Cgamma2b, Cgamma2a, Cepsilon, and Calpha. Expression of CH genes downstream of Cdelta involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding CH gene. Previously, we demonstrated that insertion of a PGK-neor cassette at two sites downstream of Calpha inhibits, in cultured B cells, GT of and CSR to a subset of CH genes (including Cgamma3, Cgamma2a, Cgamma2b, and Cepsilon) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neor cassette in place of sequences in the Igamma2b locus inhibits GT of and CSR to the upstream Cgamma3 gene, but has no major effect on the downstream Cgamma2a and Cepsilon genes. Moreover, replacement of the Cepsilon exons with a PGK-neor cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream Cgamma3, Cgamma2b, and Cgamma2a genes. As with the PGK-neor insertions 3' of Calpha studied previously, the Cgamma1 and Calpha genes were less affected by these mutations both in culture and in mice, whereas the Cgamma2b gene appeared less affected in vivo. Our findings support the existence of a long-range 3' IgH regulatory region required for GT of and CSR to multiple CH genes and suggest that PGK-neor cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent CH genes upstream of the insertion.

During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression. For this purpose, we introduced an activated Ras expression construct into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes and assayed differentiation potential by recombination activating gene (RAG) 2-deficient blastocyst complementation. We found that activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by mu HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of kappa LC gene rearrangement and a preference for kappa over lambda LC gene rearrangement similar to that of wild-type B cells. We discuss these findings in the context of potential mechanisms that may regulate Ig LC gene rearrangement.

The intronic Ig heavy chain (IgH) enhancer, which consists of the core enhancer flanked by 5' and 3' matrix attachment regions, has been implicated in control of IgH locus recombination and transcription. To elucidate the regulatory functions of the core enhancer and its associated matrix attachment regions in the endogenous IgH locus, we have introduced targeted deletions of these elements, both individually and in combination, into an IgHa/b-heterozygous embryonic stem cell line. These embryonic stem cells were used to generate chimeric mice by recombination activating gene-2 (Rag-2)-deficient blastocyst complementation, and the effects of the introduced mutations were assayed in mutant B cells. We find that the core enhancer is necessary and sufficient to promote normal variable (V), diversity (D), and joining (J) segment recombination in developing B lineage cells and IgH locus transcription in mature B cells. Surprisingly, the 5' and 3' matrix attachment regions were dispensable for these processes.

Germline CH transcripts initiate from a promoter upstream of a non-coding I exon, proceed through the switch (S) region and terminate downstream of the associated CH exons. To elucidate the role of germline transcription in Ig heavy chain class switch recombination (CSR), we used gene targeting in embryonic stem (ES) cells to replace most of the Igamma2b exon from immediately 3' of the majority of transcription initiation sites to beyond its donor splice site with a PGK-neo(r)gene inserted in the same transcriptional orientation as the endogenous unit. The mutation was introduced into both alleles of ES cell lines (referred to as gamma2-b(N/N)) and the neo(r) gene was deleted (referred to as Igamma2b-/-) by the loxP/Cre method. These mutations were assayed for effects on CSR in B cells derived via RAG-2-deficient blastocyst complementation. Igamma2b-/- B cells lacked ability to switch to IgG2b both in vivo and in vitro, and, correspondingly, showed no germline transcription through the Igamma2b exon, Sgamma2b or the Cgamma2b region. In contrast, Igamma2b(N/N) B cells switched at normal levels to IgG2b and showed substantial transcription through the Sgamma2b and Cgamma2b regions. Taken together, these results show that the Igamma2b sequences, per se, are not necessary for mediating CSR since a transcribed PGK-neo(r) gene can replace its function. However, the deleted portion of the Igamma2b exon and splice donor site apparently contain sequences necessary for efficient germline gene transcription and thus for CSR to IgG2b.

The 40-kb region downstream of the most 3' immunoglobulin (Ig) heavy chain constant region gene (Calpha) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5' enhancers in this cluster (respectively referred to as HS3a and HS1,2) were replaced either with a pgk-neor cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3aDelta and HS1,2Delta, respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis-acting, suggested that inhibition might result from pgk-neor cassette gene insertion rather than enhancer deletion. Accordingly, CSR returned to normal in B cells homozygous for the HS3aDelta or HS1,2Delta mutations. In addition, induced expression of the specifically targeted pgk-neor genes was regulated similarly to that of germline CH genes. Our findings implicate a 3' CSR regulatory locus that appears remarkably similar in organization and function to the beta-globin gene 5' LCR and which we propose may regulate differential CSR via a promoter competition mechanism.

The IgH locus intronic enhancer (Emu), located in the intron between the J(H) segments and the Cmu gene, and flanked by two matrix attachment regions (MAR), has been shown to be a major regulator of IgH gene transcription and VDJ recombination. To define the potential role of Emu plus MAR in class switch recombination (CSR), we generated IgG-expressing hybridomas from B cells heterozygous for mutations that delete all of these elements or replace them with a neo(r) gene and analyzed the switch status of the mutated IgH loci. Emu/MAR-deleted IgH loci displayed a highly significant, although not complete, decrease in CSR when compared to unmutated loci in normal hybridomas. Surprisingly, mutant loci with a pgk promoter-driven neo(r) gene replacing the Emu/MAR showed relatively normal switch frequency. These findings indicate that the Emu/MAR region plays a significant, but not necessary role in facilitating class switching at the mu locus. Potential mechanisms for these findings are discussed.

To investigate potential roles of the RAG-1 and RAG-2 gene products in Ig heavy chain class recombination (CSR), we have generated RAG-1(-/-) and RAG-2(-/-) mice which contain both a rearranged Ig HC V(D)J gene (referred to as B1-8) inserted into the endogenous Ig heavy chain (HC) locus in place of the JH segments, and a rearranged lambda1 light chain (LC) transgene (which are referred to as RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice respectively). The B1-8 HC gene and lambda LC genes encode proteins that associate to form a complete Ig molecule, the expression of which leads to substantial reconstitution of the peripheral B cell compartments of RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice. Both RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda mice have relatively normal levels of the various IgG isotypes, but greatly reduced levels of serum IgM and IgA compared to normal littermates. Furthermore, RAG-1(-/-)B1-8lambda and RAG-2(-/-)B1-8lambda B cells activated in vitro with lipopolysaccharide (LPS) or LPS plus IL-4 responded similarly to control B cells with respect to surface expression and secretion of IgG3, IgG1, IgG2b, IgG2a and IgE, but again were deficient in the secretion of IgM. Together, these findings indicate that neither RAG-1 nor RAG-2 expression is required for efficient class switching to most HC isotypes in B cells.

To test for effects on B cell differentiation, we introduced immunoglobulin mu heavy chain (HC) and Bcl-2 transgenes, separately or together, into recombination-activating gene 2 (RAG-2)-deficient mice. Transgenic Bcl-2 expression led to increased numbers of RAG-deficient pro-B cells, but did not promote their further differentiation. Expression of the mu HC transgene promoted the differentiation of RAG-deficient pro-B cells into pre-B cells that also expressed certain differentiation markers characteristic of even more mature B cell stages. However, the extent of the mu HC-dependent differentiation effects was greatly enhanced by coexpression of the transgenic Bcl-2 gene, and a subset of pre-B cells from both HC and HC, Bcl-2-transgenic RAG-2-deficient animals expressed surface mu HCs that were functional as judged by cross-linking experiments. These experiments demonstrate that the pro-B to pre-B transition in vivo cannot be effected by the expression of Bcl-2 alone, and that nontransformed immature B-lineage cells are competent to receive signals through a surface mu complex.

We replaced the IgH 3' enhancer (3'EH) region with a neomycin resistance gene in ES cells and generated chimeric mice in which all mature lymphocytes were either heterozygous (3'EH+/-) or homozygous (3'EH-/-) for the mutation. In vitro activated 3'EH-/- B cells responded similarly to 3'EH+/- B cells with respect to proliferation and secretion of IgM and IgG1 but were specifically deficient in IgG2a, IgG2b, IgG3, and IgE secretion. These isotype deficiencies correlated with a deficiency in accumulation of transcripts from and class switching to affected CH genes. In vivo, chimeric mice containing only 3'EH-/- B cells were deficient in serum IgG2a and IgG3. We propose that the 3'EH-/- mutation disrupts the activity of a regulatory region that influences heavy chain class switching to several different CH genes that lie as far as 100 kb upstream of the mutation.

Germline CH transcripts initiate from a non-coding I exon and terminate downstream of the associated CH exons. Ig heavy chain class switch recombination from the VDJ-C mu gene to particular downstream CH genes appears to be regulated by a process that involves mitogen and/or cytokine induction of germline CH transcripts from the downstream genes. We have examined the expression of germline C mu transcripts (I mu-C mu transcripts) in splenic B cells and pre-B cell lines after cytokine and mitogen stimulation. In contrast to the expression of the germline transcripts from downstream CH genes, expression of germline C mu transcripts was constitutive and unaffected by mitogen and cytokine treatment. After a primary switch recombination event, the germline I mu promoter, which is now associated with a downstream CH gene, continues to be active--leading to the generation of a novel germline transcript consisting of the I mu exon spliced to the CH exons of the switched CH gene. We discuss the potential role of the expression of hybrid I mu-containing transcripts in the class switch process. We also describe a novel and sensitive assay, based on the detection of the hybrid I mu-containing transcripts, that allows detection of class switch recombination events even in heterogeneous populations of cells.

To investigate the role of intronic immunoglobulin heavy chain (IgH) enhancer (E mu) in generating accessibility of the JH locus for VDJ recombination, we generated ES cells in which E mu or its flanking sequences were mutated by replacement with or insertion of an expressed neor gene. Heterozygous mutant ES cells were used to generate chimeric mice from which pre-B cell lines were derived by transformation of bone marrow cells with Abelson murine leukemia virus (A-MuLV). Comparison of the rearrangement status of the normal and mutated alleles in individual pre-B cell lines allowed us to assay for cis-acting effects of the mutations. Replacement of a 700 bp region immediately downstream from the core E mu [which includes part of the 3' matrix associated region (MAR) and the I mu exon] had no obvious effect on rearrangement of the targeted allele, indicating that insertion of a transcribed neor gene into the JH-C mu intron does not affect JH accessibility. In contrast, replacement of an overlapping 1 kb DNA fragment that contains the E mu resulted in a dramatic cis-acting inhibition of rearrangement, demethylation and germline transcription of the associated JH locus. Surprisingly, insertion of the neor gene into the 5' MAR sequence approximately 100 bp upstream of the core E mu also dramatically decreased recombination of the linked JH locus; but, in many lines, did not prevent demethylation of this locus. We conclude that integrity of the E mu and upstream flanking sequences is required for efficient rearrangement of the JH locus and that demethylation of this locus, per se, does not necessarily make it a good substrate for VDJ recombination.

LPS stimulation of B lymphocytes induces germline transcription of and subsequent switching to the gamma 2b gene. Mature germline transcripts contain an I exon (non-coding) spliced to the C gamma 2b exons. To investigate the role of germline transcription and/or transcripts in heavy chain class switching, we have replaced the germline I gamma 2b promoter and I exon in ES cells with an expressed neomycin resistance gene. The mutated chromosome retains the downstream target sequence for switch recombination (S regions) and all sequences necessary for expression of a switched gamma 2b gene. Wild-type or mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all lymphocytes were ES-cell derived. Chimeras derived from injection of heterozygous mutant ES cells had normal levels of serum IgG2b, but their splenic B cells showed a partial decrease in ability to switch to gamma 2b. Strikingly, B lymphocytes from chimeras derived by injection of homozygous mutant ES cells were deficient in IgG2b production both in vivo and in vitro, but normal with respect to production of other Ig heavy chain isotypes. Additional studies demonstrated that lack of ability to produce IgG2b by the mutant B cells correlated with lack of germline transcription and resulted from a specific defect in class-switch recombination to S gamma 2b. Together, these studies demonstrate that the I region is an important regulatory element for control of class-switch recombination.

Recent work has shown that the ability of cytokines to direct immunoglobulin heavy chain class-switch recombination to particular heavy chain constant (C) region (CH) genes correlates with the induction of specific germ-line CH transcripts. To test the role of germ-line transcripts in class switching, we have used homologous recombination to mutate the immunoglobulin heavy chain locus of the 18.81A20 murine pre-B-cell line. In the parent cell line, the combination of interleukin-4 (IL-4) and lipopolysaccharide (LPS) induces germ-line epsilon locus transcription prior to class switching to epsilon. The heavy chain locus of the mutated cell line contains the immunoglobulin heavy chain enhancer and variable region gene promoter in place of the LPS/IL-4-responsive germ-line epsilon promoter. The mutant cell line constitutively transcribes the epsilon locus in the absence of IL-4. Strikingly, the mutant cell line also switches to epsilon in the absence of IL-4. This result demonstrates that, at least in the 18.81A20 cell line, germ-line epsilon transcription plays a direct role in class switching to the epsilon locus. In addition, the ability to change the pattern of class switching by altering transcriptional activity indicates that transcription of germ-line CH is mechanistically important in regulation of class switching.

Transgenic mice that contain constructs of the L-myc gene under the transcriptional control of the immunoglobulin heavy chain enhancer (E mu) develop thymic hyperplasia and are predisposed to T cell lymphomas. Here we describe a second form of malignancy that occurs in aging E mu L-myc transgenic mice. The mean latency period for the development of this malignancy is longer compared with the E mu L-myc T cell lymphomas but the overall incidence is increased threefold. The histopathological morphology is that of a highly malignant mesenchymal neoplasm that closely resembles human fibrous histiocytoma. The tumor cells were classified as myelomonocytic on the basis of several lineage-specific markers and the lack of rearrangements of the immunoglobulin heavy chain and the T cell receptor beta loci. Cultured tumor cells produce macrophage colony-stimulating factor (M-CSF) protein and express the M-CSF receptor, suggesting the involvement of an autocrine loop in this malignancy. Similar to the E mu L-myc T cell lymphomas, these tumors show high-level transgene expression but no detectable levels of endogenous c-myc mRNA, directly implicating the deregulated expression of L-myc in the generation of this malignancy. E mu L-myc myelomonocytic tumors show consistent trisomy of chromosome 16, implicating this as a secondary event in the development of this tumor. In the light of recent findings that L-myc is expressed in human myeloid leukemias and in several human myeloid tumor cell lines, the results described here might implicate L-myc in the development of naturally occurring myeloid neoplasias.

X-linked agammaglobulinemia (XLA) is characterized by an arrest in early B lymphocyte differentiation. Precursor B cells are present in the bone marrow (BM), whereas peripheral blood B cell numbers are severely decreased. A series of Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) was established from peripheral blood of three XLA patients belonging to one pedigree. These BLCL manifested productive VHDJH rearrangements and a random utilization of the VH families. The CDR3 regions of the rearrangements varied in length from 12 to 47 nucleotides and included N regions in all cases. The results supported the conclusion that the few B lymphocytes in peripheral blood of XLA patients exhibit all mechanisms that generate immunoglobulin (Ig) heavy (H) chain diversity. However, no evidence for somatic mutation was found. Within the VH3 family 50% of the expressed VH gene segments belonged to a single subgroup and within the VH4 family a preferential utilization of one VH4 gene element was observed. The utilization of H chain joining (HH) elements was biased to JH4 and JH6 and a high percentage of the CDR3 regions was found to be generated by unconventional mechanisms, such as multiple D usage and the fusion of D elements to D segments with irregular recombination recognition signals. These unique features of the recombined and expressed VHDJH regions in XLA may explain the inability of XLA patients to respond to a variety of antigens. Alternatively, they could be secondary to a B lymphocyte maturation defect in XLA.

The human VH5 family consists of two functional genes and one pseudogene. We have found a novel 1.2-kb VH5 gene transcript in normal fetal liver and cord blood and in transformed B lineage cells. VH5-positive cDNA clones were isolated from precursor B acute lymphoblastic leukemia, B chronic lymphoblastic leukemia, Epstein-Barr Virus-transformed B cell lines, and cord blood, and were identified as transcripts of unrearranged VH5 genes (germline transcripts). The cDNA clones were derived from both functional and pseudo-VH5 genes. Most germline transcripts appear to initiate at the normal VH promoter and are cleaved and polyadenylated at sites several hundred bases downstream of the VH5 coding region. Correct splicing of the leader intron was observed in all clones. In functional and pseudo-VH5 cDNAs, an open translational reading frame extends from the leader to a termination codon in the nonamer. Only limited polymorphisms were observed in the coding as well as flanking regions of the VH5 transcripts. Functional and pseudo-VH5 transcripts and previously identified murine germline VHJ558 transcripts are discussed.

Preferential usage of JH-proximal VH genes has been demonstrated in immature murine B cell repertoires. To determine whether this phenomenon is also evident in human repertoires, we studied utilization of VH6, the most JH-proximal human VH gene. Examination of VH gene usage in a panel of precursor B cell acute lymphoblastic leukemia samples indicated that 15% of the IgH rearrangements utilized VH6. VH6 is a single-member family in a total repertoire of 100-200 VH genes; thus, if usage were purely random, one would expect VH6 rearrangement frequency to be less than 1%. Analysis of VH gene usage in normal lymphoid tissues also revealed biased usage of VH6. VH6 was preferentially utilized in 16- to 24-week-old fetal liver as compared to adult peripheral blood mononuclear cells or spleen. Possible implications of the conservation of preferential usage of JH-proximal genes in both immature murine and human repertoires are discussed.

We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.

A cDNA clone that encodes the heavy chain variable region (VH) of an IgM M-protein with anti-myelin-associated glycoprotein (MAG) activity secreted by chronic lymphocytic leukemia cells (B-C11) from a patient with peripheral neuropathy was cloned and sequenced. The JH region was identical to the germline JH4 sequence except for deletion of a thymidine residue at the site of D-JH recombination, and the D region showed greatest homology to DM2. Sequence analysis of the VH region revealed greatest homology to VH26, a member of the VH3 gene family, but homology was only 83.7% over 326 bases, suggesting that it was derived from as yet an unidentified member of the VH3 gene family.

Elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire remains a major challenge in immunology. Knowledge of V gene diversity, organization, and expression is important to an understanding of the formation of the antibody repertoire in normal as well as diseased states. In the last few years, great advances have been made in our understanding of the human heavy chain variable region (VH) gene locus. In this review we present the current knowledge of VH gene diversity, organization, and utilization in normal individuals followed by a discussion of the possible relevance of these findings to autoimmunity.

A cell line that switches from mu to gamma 2b synthesis during growth in culture uses the same VH region for both heavy chains but retains two copies of the Cmu gene. This suggests that the mu to gamma 2b class switch can occur, at least in part, by an RNA processing mechanism. Regulatory variants of this cell line lose constitutive mu-chain synthesis but simultaneously acquire lipopolysaccharide (LPS)-inducible synthesis of that chain. This co-variation is allele-specific and is correlated to a large deletion of DNA in the JH--Cmu intron.

Immunoglobulin (Ig) gene expression has been followed during the later stages of development of the murine fetal liver. Biosynthetic labeling and immunoprecipitation were used to isolate Ig-related polypeptides from fetal and neonatal livers. By examination of the specific immune precipitates, the earliest detectable Ig was shown to consist only of mu heavy chain. At about the time of birth, when light chain synthesis became evident, separation of surface Ig-positive cells from surface Ig-negative cells by using anti-Ig-coated dishes showed that cells lacking surface Ig (pre-B lymphocytes) synthesized only mu chains. Thus, commencement of light chain synthesis was closely coordinated with the appearance of surface Ig. Ig RNA species were examined by electrophoretic fractionation and hybridization with cloned Ig DNA sequences. The sizes and amounts of Ig mRNA were found to correlate with the pattern of mu and light chain protein biosynthesis. mu chain RNA species appeared earlier in gestation than light chain RNA did, and only after birth did light chain sequences reach levels equivalent to those of mu chain. Cell populations enriched in pre-B lymphocytes also contained an excess of mu over light chain mRNA.

We investigated the immunochemical and molecular characteristics of murine monoclonal rheumatoid factors. Study of the fine specificity of 20 monoclonal rheumatoid factor antibodies shows a wide degree of heterogeneity. However, many express an interstrain cross-reactive idiotype. We show that our rheumatoid factors utilize a restricted set of the heavy-chain variable region (VH) repertoire representing the more 3' VH families. Preferential expression of 3' VH families is known to occur early in development. We report the nucleotide sequence of two cloned rheumatoid factor VH genes, Y19-10 (VH J558) and 129-48 (VH 7183) in which no major differences are observed between VH genes encoding the heavy chain of autoantibodies and antibodies against foreign antigens.

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.