AID- and Ung-dependent generation of staggered double-strand DNA breaks in immunoglobulin class switch DNA recombination: a post-cleavage role for AID

Class switch DNA recombination (CSR) substitutes an immunoglobulin (Ig) constant heavy chain (C(H)) region with a different C(H) region, thereby endowing an antibody with different biological effector functions. CSR requires activation-induced cytidine deaminase (AID) and occurrence of double-strand DNA breaks (DSBs) in S regions of upstream and downstream C(H) region genes. DSBs are critical for CSR and would be generated through deamination of dC by AID, subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and nicking by apurinic/apyrimidic endonuclease (APE) of nearby abasic sites on opposite DNA strands. We show here that in human and mouse B cells, S region DSBs can be generated in an AID- and Ung-independent fashion. These DSBs are blunt and 5'-phosphorylated. In B cells undergoing CSR, blunt and 5'-phosphorylated DSBs are processed in an AID- and Ung-dependent fashion to yield staggered DNA ends. Blunt and 5'-phosphorylated DSBs can be readily detected in human and mouse AID- or Ung-deficient B cells. These B cells are CSR defective, but show evidence of intra-S region recombination. Forced expression of AID in AID-negative B cells converts blunt S region DSBs to staggered DSBs. Conversely, forced expression of dominant negative AID or inhibition of Ung by Ung inhibitor (Ugi) in switching B cells abrogates the emergence of staggered DSBs and concomitant CSR. Thus, AID and Ung generate staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends, whose generation is AID- and Ung-independent, thereby outlining a post-cleavage role for AID in CSR.     

The role of activation-induced deaminase in antibody diversification and genomic instability

More than a decade ago, activation-induced deaminase (AID) was identified as the initiator for somatic hypermutation (SHM) and class switch recombination (CSR). Since then, tremendous progress has been achieved toward elucidating how AID functions. AID targets the highly repetitive switch regions of the immunoglobulin heavy chain (IgH) locus to induce DNA double-strand breaks (DSBs), which can be rejoined, leading to switch of constant regions of antibody. When targeting to variable region exons of IgH and IgL loci, AID predominantly induces point mutations, termed SHM, resulting in increased affinity of antibody for antigen. While SHM and CSR enhance antibody diversity, AID-initiated DSBs and mutations may predispose B cells to carcinogenesis. This review focuses on the mechanisms that provide the specificity of AID targeting to Ig loci and the role of AID in genomic instability.     

Analysis of class switch recombination and somatic hypermutation in patients affected with autosomal dominant hyper-IgM syndrome type 2

Autosomal recessive form of hyper-IgM syndrome type 2 (AR-HIGM2) is secondary to mutations affecting both alleles of AICDA gene encoding activation-induced cytidine deaminase, characterized by defects of immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in most of the patients. We herein report the immunological phenotype of seven patients carrying a single heterozygous R190X mutation in AICDA. Variable defect in in vivo CSR inherited as an autosomal dominant (AD) trait strongly suggests that this heterozygous AICDA mutation causes HIGM (AD-HIGM2). In AD-HIGM2 B cells, CSR was consistently found impaired in vitro. However, in contrast to AR-HIGM2, the CSR-induced double-stranded DNA breaks in the switch region of IgM heavy chain gene were detected. The SHM frequency in V regions of IgM heavy chain gene in B cells was normal in all (but one patient). The characteristics of the AD-HIGM2 phenotype indicate that the AID C-terminal region may be involved in DNA repair machinery required for CSR.     

Use of a simple,general targeting vector for replacing the DNA of the heavy chain constant region in mouse hybridoma cells

It is often necessary to modify the constant region of the immunoglobulin (Ig) heavy chain in order to produce Ig with optimal properties. In the case of Ig production by mouse hybridoma cells, it is possible to modify the Ig heavy chain (IgH) locus by gene targeting to achieve the desired changes. DNA segments from the JH-S micro region and from the region 3' of Calpha are normally present in the functional IgH gene of all hybridomas, regardless of the heavy chain class which is expressed. Consequently, these DNA segments could in principle serve as 5' and 3' homology regions to create a "universal" targeting vector for replacing the constant region exons in the IgH locus of any hybridoma cell. The practicality of this vector design has been uncertain. That is, the extent of the chromosomal DNA which would be replaced by a universal targeting vector would be as little as 5 kb (in a cell producing the alpha heavy chain) and as much as 180 kb (in a micro -producing cell), and it has been uncertain whether it would be practical to generate such long chromosomal deletions by gene targeting. Using a vector of this design, we found (a) that correctly targeted recombinant cells lacking the 180 kb DNA segment occurred at a low but usable frequency, (b) that these recombinants expressed the modified IgH locus at the same rate as the original hybridoma and (c) that IgH expression in these cell lines was stable. Our results thus indicate that this vector design is suitable for modifying IgH loci expressing any heavy chain, provided that an efficient selection or screening for targeted recombinants is available.     

High-level expression of a human immunoglobulin gamma 1 transgene depends on switch region sequences.

We describe that chimeric mouse-human immunoglobulin heavy chain (IgH) genes lacking a switch region and controlled by an IgH promoter and the intronic enhancer are only weakly expressed in transgenic mice. Insertion of part of the human C gamma 1 or murine Cmu switch region into the major intron of the chimeric IgH gene results in a 10(2)-to 10(3)-fold increase in transgene expression. Analysis of B cell hybridoma clones from transgenic mice suggests that switch sequences influence IgH transgene expression at the cellular level. However, the effect of switch region sequences on IgH gene expression observed in vivo is not apparent in transfected B cell lines. These results indicate that switch region sequences which are located proximal to the constant part of the gene and which are normally present in a rearranged IgH gene after class switching represent a novel type of regulatory element that plays a critical role in IgH gene expression in vivo.     

Production of heavy-chain class-switch variants of human monoclonal antibody by recombinant DNA technology

We have previously established a human-mouse heterohybridoma (H6-3C4), which produced a human sperm-immobilizing antibody (mu, lambda of human type). The human rearranged immunoglobulin mu-chain and lambda-chain genes were cloned from the hybridoma H6-3C4. The cloned V region of the heavy chain (VH) gene was ligated to human immunoglobulin gamma 1-heavy chain constant region (C gamma 1) genes. This resulted in the heavy-chain class-switch from mu-chain to gamma 1-chain of H6-3C4 antibody. The class-switched heavy-chain gene as well as the cloned lambda-chain gene were introduced into mouse myeloma cell line X63Ag8.653 by protoplast fusion and electroporation. The stable transformants produced the human IgG monoclonal antibody, which fully retained specificity to human sperm cells and sperm-immobilizing activity.     

Immunoglobulin gene conversion: insights from bursal B cells and the DT40 cell line.

Chicken B cells diversify their immunoglobulin (Ig) light and heavy chain genes by pseudogene templated gene conversion within the bursa of Fabricius. Although Ig gene conversion was initially believed to occur only in birds, it is now clear that most farm animals also use this elegant mechanism to develop an immunoglobulin gene repertoire. The best model to study Ig gene conversion remains the chicken Ig light chain locus due to its compact size and the fact that all the pseudogene donors are sequenced. Furthermore, gene conversion continues in the bursa-derived DT40 cell line whose genome can be easily modified by targeted integration of transfected constructs. Disruption of the AID gene, which had been shown to control somatic hypermutation and switch recombination in mammals leads to a complete block of gene conversion in DT40 indicating that all B-cell specific repertoire formation is controlled by the same gene. Here, we review the genetics and the molecular mechanism of Ig gene conversion based on sequence analysis of bursal B cells and gene disruption studies in the DT40 cell line.     

The molecular events in heavy chain class-switching

Heavy chain class-switching is the process by which B lymphoid cells change the constant region of the immunoglobulin heavy chain they produce. Class-switching is most commonly accomplished by recombinationldeletion between switch recombination regions that lie upstream of each germline heavy chain constant region gene. Recent studies support a model that recombination to specific switch regions is directed by modulation of the accessibility of these regions to a common class-switch recombination system. T cell lymphokines seem to be able to alter the accessibility of different heavy chain constant region loci, and thereby direct the specificity of class-switch recombination in B cells.     

Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector

Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two-sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross-over takes place; (ii) a 5′ flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3′ flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3′ flank is non-functional in an inverted orientation.     

Expression of genetically engineered immunoconjugates of lymphotoxin and a chimeric anti-ganglioside GD2 antibody

Human lymphotoxin was genetically conjugated to the constant region of a human gamma 1 immunoglobulin gene at the end of either the second (CH2-LT) or third (CH3-LT) constant region domain. The altered heavy chain constant regions were combined in a plasmid vector together with the variable regions of a mouse anti-ganglioside GD2 antibody 14.18 and the human kappa constant region. The resulting immunoconjugate constructs were expressed in transfected hybridoma cells and tested for both their antibody and lymphotoxin activities. The two constructs were assembled to varying degrees depending on whether the third heavy chain constant region was present. Both forms retained their ability to bind antigen and mediate ADCC but only CH3-LT was able to mediate the lysis of melanoma target cells in the presence of human complement. Lymphotoxin activity, as defined in a cytolytic assay with mouse fibroblasts, was found to increase significantly as a function of heavy chain assembly and to be equivalent to unconjugated lymphotoxin. Neither of the constructs were cytotoxic for antigen-bearing melanoma cells that are normally resistant to lymphotoxin and tumor necrosis factor alpha. Such immunoconjugates may prove useful in targeting cytokines to the site of antigen-bearing cells in vivo. In this case, as a means of eliciting an inflammatory response at the site of a solid tumor.     

Identification of an AID-independent pathway for chromosomal translocations between the Igh switch region and Myc

Chromosomal translocations involving immunoglobulin heavy chain (Igh) switch regions and an oncogene such as Myc represent initiating events in the development of many B cell malignancies. These translocations are widely thought to result from aberrant class-switch recombination. To test this model, we measured translocations in mice deficient in activation-induced cytidine deaminase (AID) that lack class-switch recombination. We found that AID made no measurable contribution to the generation of initial translocations, indicating that the intrinsic fragility of the switch regions or a pathway unrelated to AID is responsible for these translocations. In contrast, the outgrowth of translocation-positive cells was dependent on AID, raising the possibility that AID is important in tumor progression, perhaps by virtue of its mutagenic properties.     

The splicing regulator PTBP2 interacts with the cytidine deaminase AID and promotes binding of AID to switch-region DNA

During immunoglobulin class-switch recombination (CSR), the cytidine deaminase AID induces double-strand breaks into transcribed, repetitive DNA elements called switch sequences. The mechanism that promotes the binding of AID specifically to switch regions remains to be elucidated. Here we used a proteomic screen with in vivo biotinylation of AID to identify the splicing regulator PTBP2 as a protein that interacts with AID. Knockdown of PTBP2 mediated by short hairpin RNA in B cells led to a decrease in binding of AID to transcribed switch regions, which resulted in considerable impairment of CSR. PTBP2 is thus an effector of CSR that promotes the binding of AID to switch-region DNA.     

抗人CD16单克隆抗体可变区基因的克隆和表达

目的：克隆抗人CD16单克隆抗体重，轻链可变区（VH，VL）基因并合成单链抗体（ScFv)基因。方法：从分泌抗人CD16单克隆抗体的杂交瘤细胞B88－9中提取总RNA，应用RT－PCR技术获得抗CD16单克隆抗体的VH，VL基因，用连接肽（Linker)肽VH和VL连接成具有VH－Linker-VL结构的ScFv基因，将其克隆到表达载体pcDNA3.1( )，并转杂COS－7细胞。结果：VH基因长度为354bp，属于鼠抗体可变区重链基因家族I（B）亚群，VL基因长度为333bp，属于鼠抗体可变区kappa轻链基因家族Ⅲ亚群，采用夹心ELISA方法检测到ScFv的表达。结论：抗人CD16单克隆抗体VH与VL基因的克隆和ScFv基因的构建为基于CD16的导向免疫治疗奠定了基础。     

Class switch recombination: a friend and a foe

The effector functions of the antibody are determined by the heavy chain constant region (CH). During CSR the primarily expressed mu constant region (Cmu) of the heavy chain is replaced with a downstream isotype Cgamma, Calpha or Cepsilon. The murine immunoglobulin heavy chain (IgH) locus contains eight different CH genes. Class switch recombination (CSR) involves a recombination between two different repetitive switch (S) region sequences, located upstream of each CH gene and the deletion of the intervening DNA. However, this protecting mechanism is also involved in aberrant chromosomal translocations and generation of B cell malignancies. It is also involved in susceptibility to autoimmunity. The current review focuses on the basic mechanism of CSR and the adverse outcomes that it may cause.     

AID mutates a non-immunoglobulin transgene independent of chromosomal position

It is unknown how activation-induced cytidine deaminase (AID) targets immunoglobulin (Ig) genes during somatic hypermutation. Results to date are difficult to interpret: while some results argue that Ig genes have special sequences that mobilize AID, other work shows that non-Ig transgenes mutate. In this report, we have examined the effects of the intronic mu enhancer on the somatic hypermutation rates of a retroviral vector. For this analysis, we used centroblast-like Ramos cells to capture as much of the natural process as possible, used AIDhi and AIDlow Ramos variants to ensure that mutations are AID induced, and measured mutation of a GFP-provirus to achieve greater sensitivity. We found that mutation rates of the non-Ig provirus were AID-dependent, were similar at different genomic loci, but were approximately 10-fold lower than the V-region suggesting that AID can mutate non-Ig genes at low rates. However, the intronic mu enhancer did not increase the mutation rates of the provirus. Interestingly, exogenous over-expression of AID revealed that the V-region mutation rate can be saturated by lower levels of AID than the provirus, suggesting that selective mutation of Ig sequences is compromised in cells that over-express AID.