Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice.

DNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here, we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1-/- and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1-/- mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1-/- mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1-/- mice was comparably defective, switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1-/- mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1-/- mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.

The role of HIRA-dependent H3.3 deposition and its modifications in the somatic hypermutation of immunoglobulin variable regions.

The H3.3 histone variant and its chaperone HIRA are involved in active transcription, but their detailed roles in regulating somatic hypermutation (SHM) of immunoglobulin variable regions in human B cells are not yet fully understood. In this study, we show that the knockout (KO) of HIRA significantly decreased SHM and changed the mutation pattern of the variable region of the immunoglobulin heavy chain (IgH) in the human Ramos B cell line without changing the levels of activation-induced deaminase and other major proteins known to be involved in SHM. Except for H3K79me2/3 and Spt5, many factors related to active transcription, including H3.3, were substantively decreased in HIRA KO cells, and this was accompanied by decreased nascent transcription in the IgH locus. The abundance of ZMYND11 that specifically binds to H3.3K36me3 on the IgH locus was also reduced in the HIRA KO. Somewhat surprisingly, HIRA loss increased the chromatin accessibility of the IgH V region locus. Furthermore, stable expression of ectopic H3.3G34V and H3.3G34R mutants that inhibit both the trimethylation of H3.3K36 and the recruitment of ZMYND11 significantly reduced SHM in Ramos cells, while the H3.3K79M did not. Consistent with the HIRA KO, the H3.3G34V mutant also decreased the occupancy of various elongation factors and of ZMYND11 on the IgH variable and downstream switching regions. Our results reveal an unrecognized role of HIRA and the H3.3K36me3 modification in SHM and extend our knowledge of how transcription-associated chromatin structure and accessibility contribute to SHM in human B cells.

A Bayesian model based computational analysis of the relationship between bisulfite accessible single-stranded DNA in chromatin and somatic hypermutation of immunoglobulin genes.

The B cells in our body generate protective antibodies by introducing somatic hypermutations (SHM) into the variable region of immunoglobulin genes (IgVs). The mutations are generated by activation induced deaminase (AID) that converts cytosine to uracil in single stranded DNA (ssDNA) generated during transcription. Attempts have been made to correlate SHM with ssDNA using bisulfite to chemically convert cytosines that are accessible in the intact chromatin of mutating B cells. These studies have been complicated by using different definitions of "bisulfite accessible regions" (BARs). Recently, deep-sequencing has provided much larger datasets of such regions but computational methods are needed to enable this analysis. Here we leveraged the deep-sequencing approach with unique molecular identifiers and developed a novel Hidden Markov Model based Bayesian Segmentation algorithm to characterize the ssDNA regions in the IGHV4-34 gene of the human Ramos B cell line. Combining hierarchical clustering and our new Bayesian model, we identified recurrent BARs in certain subregions of both top and bottom strands of this gene. Using this new system, the average size of BARs is about 15 bp. We also identified potential G-quadruplex DNA structures in this gene and found that the BARs co-locate with G-quadruplex structures in the opposite strand. Using various correlation analyses, there is not a direct site-to-site relationship between the bisulfite accessible ssDNA and all sites of SHM but most of the highly AID mutated sites are within 15 bp of a BAR. In summary, we developed a novel platform to study single stranded DNA in chromatin at a base pair resolution that reveals potential relationships among BARs, SHM and G-quadruplexes. This platform could be applied to genome wide studies in the future.

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes.

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sµ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

AID Overlapping and Polη Hotspots Are Key Features of Evolutionary Variation Within the Human Antibody Heavy Chain (IGHV) Genes.

Somatic hypermutation (SHM) of the immunoglobulin variable (IgV) loci is a key process in antibody affinity maturation. The enzyme activation-induced deaminase (AID), initiates SHM by creating C → U mismatches on single-stranded DNA (ssDNA). AID has preferential hotspot motif targets in the context of WRC/GYW (W = A/T, R = A/G, Y = C/T) and particularly at WGCW overlapping hotspots where hotspots appear opposite each other on both strands. Subsequent recruitment of the low-fidelity DNA repair enzyme, Polymerase eta (Polη), during mismatch repair, creates additional mutations at WA/TW sites. Although there are more than 50 functional immunoglobulin heavy chain variable (IGHV) segments in humans, the fundamental differences between these genes and their ability to respond to all possible foreign antigens is still poorly understood. To better understand this, we generated profiles of WGCW hotspots in each of the human IGHV genes and found the expected high frequency in complementarity determining regions (CDRs) that encode the antigen binding sites but also an unexpectedly high frequency of WGCW in certain framework (FW) sub-regions. Principal Components Analysis (PCA) of these overlapping AID hotspot profiles revealed that one major difference between IGHV families is the presence or absence of WGCW in a sub-region of FW3 sometimes referred to as "CDR4." Further differences between members of each family (e.g., IGHV1) are primarily determined by their WGCW densities in CDR1. We previously suggested that the co-localization of AID overlapping and Polη hotspots was associated with high mutability of certain IGHV sub-regions, such as the CDRs. To evaluate the importance of this feature, we extended the WGCW profiles, combining them with local densities of Polη (WA) hotspots, thus describing the co-localization of both types of hotspots across all IGHV genes. We also verified that co-localization is associated with higher mutability. PCA of the co-localization profiles showed CDR1 and CDR2 as being the main contributors to variance among IGHV genes, consistent with the importance of these sub-regions in antigen binding. Our results suggest that AID overlapping (WGCW) hotspots alone or in conjunction with Polη (WA/TW) hotspots are key features of evolutionary variation between IGHV genes.

CLL intraclonal fractions exhibit established and recently acquired patterns of DNA methylation.

Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

Human Immunodeficiency Virus Tat Protein Aids V Region Somatic Hypermutation in Human B Cells.

Long-term survivors of human immunodeficiency virus (HIV) infection have been shown to have a greatly increased incidence of B cell lymphomas. This increased lymphomagenesis suggests some link between HIV infection and the destabilization of the host B cell genome, a phenomenon also suggested by the extraordinary high frequency of mutation, insertion, and deletion in the broadly neutralizing HIV antibodies. Since HIV does not infect B cells, the molecular mechanisms of this genomic instability remain to be fully defined. Here, we demonstrate that the cell membrane-permeable HIV Tat proteins enhance activation-induced deaminase (AID)-mediated somatic hypermutation (SHM) of antibody V regions through their modulation of the endogenous polymerase II (Pol II) transcriptional process. Extremely small amounts of Tat that could come from bystander HIV-infected cells were sufficient to promote SHM. Our data suggest HIV Tat is one missing link between HIV infection and the overall B cell genomic instability in AIDS patients.IMPORTANCE Although the introduction of antiretroviral therapy (ART) has successfully controlled primary effects of human immunodeficiency virus (HIV) infection, such as HIV proliferation and HIV-induced immune deficiency, it did not eliminate the increased susceptibility of HIV-infected patients to B cell lymphomas. We find that a secreted HIV protein, Tat, enhances the intrinsic antibody diversification mechanism by increasing the AID-induced somatic mutations at the heavy-chain variable (VH) regions in human B cells. This could contribute to the high rate of mutation in the variable regions of broadly neutralizing anti-HIV antibodies and the genomewide mutations leading to B cell malignancies in HIV carriers.

Overlapping hotspots in CDRs are critical sites for V region diversification.

Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.

A source of the single-stranded DNA substrate for activation-induced deaminase during somatic hypermutation.

During somatic hypermutation (SHM), activation-induced deaminase (AID) mutates deoxycytidine on single-stranded DNA (ssDNA) generated by the transcription machinery, but the detailed mechanism remains unclear. Here we report a higher abundance of RNA polymerase II (Pol II) at the immunoglobulin heavy-chain variable (Igh-V) region compared with the constant region and partially transcribed Igh RNAs, suggesting a slower Pol II progression at Igh-V that could result in some early/premature transcription termination after prolonged pausing/stalling of Pol II. Knocking down RNA-exosome complexes, which could decrease premature transcription termination, leads to decreased SHM. Knocking down Spt5, which can augment premature transcription termination, leads to increase in both, SHM and the abundance of ssDNA substrates. Collectively, our data support the model that, following the reduction of Pol II progression (pausing or stalling) at the Igh-V, additional steps such as premature transcription termination are involved in providing ssDNA substrates for AID during SHM.

Isotype switching increases efficacy of antibody protection against staphylococcal enterotoxin B-induced lethal shock and Staphylococcus aureus sepsis in mice.

UNLABELLED: Staphylococcal enterotoxin B (SEB) is a potent toxin that is produced by Staphylococcus aureus strains and is classified as a category B select agent. We have previously shown that monoclonal antibody (MAb) 20B1, a murine anti-SEB IgG1, successfully treats SEB-induced lethal shock (SEBILS) and bacteremia that is caused by SEB-producing S. aureus. In this study, we have generated two isotype switch variants of the original IgG1 MAb 20B1, an IgG2a and IgG2b, both bearing the same variable region sequence, and compared their neutralizing and protective activity in in vitro and in vivo assays, respectively. All 3 isotypes demonstrated comparable affinity to SEB and comparable 50% inhibitory concentrations (IC50s) in T cell proliferation assays. In vivo, however, the IgG2a isotype variant of 20B1 exhibited significantly greater protection than IgG1 or IgG2b in murine SEB intoxication and S. aureus sepsis models. Protection was associated with downmodulation of inflammatory host response. Our data demonstrate that changing the isotype of already protective MAbs, without affecting their antigen specificity or sensitivity, can result in an enhancement of their protective ability. Isotype selection, therefore, should be carefully considered in the development of toxin-neutralizing MAbs and the design of antibody therapeutics. IMPORTANCE: The purpose of this study was to enhance the protective efficacy of an existing, protective monoclonal antibody against staphylococcal enterotoxin B. Using two in vivo mouse models, our study demonstrates that the protective efficacy of a monoclonal antibody may be improved by inducing an isotype switch at the Fc region of an antibody, without altering the antigen specificity or sensitivity of the antibody. The development of therapeutic MAbs with higher efficacy may allow for the achievement of equal therapeutic benefit with a lower dosage. In turn, the use of lower doses may reduce the cost of these therapies, while reducing the potential for adverse side effects.

Staphylococcal Enterotoxin B-specific monoclonal antibody 20B1 successfully treats diverse Staphylococcus aureus infections.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) has become a major health threat in the United States. Staphylococcal enterotoxin B (SEB) is a potent superantigen that contributes to its virulence. High mortality and frequent failure of therapy despite available antibiotics have stimulated research efforts to develop adjunctive therapies. METHODS: Treatment benefits of SEB-specific monoclonal antibody (mAb) 20B1 were investigated in mice in sepsis, superficial skin, and deep-tissue infection models. RESULTS: Mice challenged with a SEB-producing MRSA strain developed fatal sepsis, extensive tissue skin infection, and abscess-forming deep-seeded thigh muscle infection. Animals preimmunized against SEB or treated passively with mAb 20B1 exhibited enhanced survival in the sepsis model, whereas decrease of bacterial burden was observed in the superficial skin and deep-tissue models. mAb 20B1 bound to SEB in the infected tissue and decreased abscess formation and proinflammatory cytokine levels, lymphocyte proliferation, and neutrophil recruitment. CONCLUSIONS: mAb 20B1, an SEB-neutralizing mAb, is effective against MRSA infection. mAb 20B1 protects against lethal sepsis and reduces skin tissue invasion and deep-abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects the outcome of S. aureus infection by modulating the host's proinflammatory immune response.

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes.

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1(EK)) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1(null)) mouse. In contrast to Exo1(null/null) mice, Exo1(EK/EK) mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1(null) mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo.

HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function.

T-cell costimulation and coinhibition generated by engagement of the B7 family and their receptor CD28 family are of central importance in regulating the T-cell response, making these pathways very attractive therapeutic targets. Here we describe HERV-H LTR-associating protein 2 (HHLA2) as a member of the B7 family that shares 10-18% amino acid identity and 23-33% similarity to other human B7 proteins and phylogenetically forms a subfamily with B7x and B7-H3 within the family. HHLA2 is expressed in humans but not in mice, which is unique within the B7 and CD28 families. HHLA2 protein is constitutively expressed on the surface of human monocytes and is induced on B cells after stimulation with LPS and IFN-γ. HHLA2 does not interact with other known members of the CD28 family or the B7 family, but does bind a putative receptor that is constitutively expressed not only on resting and activated CD4 and CD8 T cells but also on antigen-presenting cells. HHLA2 inhibits proliferation of both CD4 and CD8 T cells in the presence of T-cell receptor signaling. In addition, HHLA2 significantly reduces cytokine production by T cells including IFN-γ, TNF-α, IL-5, IL-10, IL-13, IL-17A, and IL-22. Thus, we have identified a unique B7 pathway that is able to inhibit human CD4 and CD8 T-cell proliferation and cytokine production. This unique human T-cell coinhibitory pathway may afford unique strategies for the treatment of human cancers, autoimmune disorders, infection, and transplant rejection and may help to design better vaccines.

AID and Apobec3G haphazard deamination and mutational diversity.

Activation-induced deoxycytidine deaminase (AID) and Apobec 3G (Apo3G) cause mutational diversity by initiating mutations on regions of single-stranded (ss) DNA. Expressed in B cells, AID deaminates C â†’ U in actively transcribed immunoglobulin (Ig) variable and switch regions to initiate the somatic hypermutation (SHM) and class switch recombination (CSR) that are essential for antibody diversity. Apo3G expressed in T cells catalyzes C deaminations on reverse transcribed cDNA causing HIV-1 retroviral inactivation. When operating properly, AID- and Apo3G-initiated mutations boost human fitness. Yet, both enzymes are potentially powerful somatic cell "mutators". Loss of regulated expression and proper genome targeting can cause human cancer. Here, we review well-established biological roles of AID and Apo3G. We provide a synopsis of AID partnering proteins during SHM and CSR, and describe how an Apo2 crystal structure provides "surrogate" insight for AID and Apo3G biochemical behavior. However, large gaps remain in our understanding of how dC deaminases search ssDNA to identify trinucleotide motifs to deaminate. We discuss two recent methods to analyze ssDNA scanning and deamination. Apo3G scanning and deamination is visualized in real-time using single-molecule FRET, and AID deamination efficiencies are determined with a random walk analysis. AID and Apo3G encounter many candidate deamination sites while scanning ssDNA. Generating mutational diversity is a principal aim of AID and an important ancillary property of Apo3G. Success seems likely to involve hit and miss deamination motif targeting, biased strongly toward miss.

IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions.

Clonal evolution occurs during the course of chronic lymphocytic leukemia (CLL) and activation-induced deaminase (AID) could influence this process. However, this possibility has been questioned in CLL because the number of circulating AID mRNA(+) cells is exceedingly low; synthesis of AID protein by blood CLL cells has not been demonstrated; the full range of AID functions is lacking in unmutated CLL (U-CLL), and no prospective analysis linking AID expression and disease severity has been reported. The results of the present study show that circulating CLL cells and those within secondary lymphoid tissues can make AID mRNA and protein. This production is related to cell division because more AID mRNA was detected in recently divided cells and AID protein was limited to the dividing fraction and was up-regulated on induction of cell division. AID protein was functional because AID(+) dividing cells exhibited more double-stranded DNA breaks, IGH class switching, and new IGHV-D-J mutations. Each of these actions was documented in U-CLL and mutated CLL (M-CLL). Furthermore, AID protein was associated with worse patient outcome and adverse cytogenetics. We conclude that the production of fully functional AID protein by U-CLL and M-CLL cells could be involved in clonal evolution of the disease.

AIDing antibody diversity by error-prone mismatch repair.

The creation of a highly diverse antibody repertoire requires the synergistic activity of a DNA mutator, known as activation-induced deaminase (AID), coupled with an error-prone repair process that recognizes the DNA mismatch catalyzed by AID. Instead of facilitating the canonical error-free response, which generally occurs throughout the genome, DNA mismatch repair (MMR) participates in an error-prone repair mode that promotes A:T mutagenesis and double-strand breaks at the immunoglobulin (Ig) genes. As such, MMR is capable of compounding the mutation frequency of AID activity as well as broadening the spectrum of base mutations; thereby increasing the efficiency of antibody maturation. We here review the current understanding of this MMR-mediated process and describe how the MMR signaling cascade downstream of AID diverges in a locus dependent manner and even within the Ig locus itself to differentially promote somatic hypermutation (SHM) and class switch recombination (CSR) in B cells.

The ATPase activity of MLH1 is required to orchestrate DNA double-strand breaks and end processing during class switch recombination.

Antibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are similarly initiated in B cells with the generation of U:G mismatches by activation-induced cytidine deaminase but differ in their subsequent mutagenic consequences. Although SHM relies on the generation of nondeleterious point mutations, CSR depends on the production of DNA double-strand breaks (DSBs) and their adequate recombination through nonhomologous end joining (NHEJ). MLH1, an ATPase member of the mismatch repair (MMR) machinery, is emerging as a likely regulator of whether a U:G mismatch progresses toward mutation or DSB formation. We conducted experiments on cancer modeled ATPase-deficient MLH1G67R knockin mice to determine the function that the ATPase domain of MLH1 mediates in SHM and CSR. Mlh1(GR/GR) mice displayed a significant decrease in CSR, mainly attributed to a reduction in the generation of DSBs and diminished accumulation of 53BP1 at the immunoglobulin switch regions. However, SHM was normal in these mice, which distinguishes MLH1 from upstream members of the MMR pathway and suggests a very specific role of its ATPase-dependent functions during CSR. In addition, we show that the residual switching events still taking place in Mlh1(GR/GR) mice display unique features, suggesting a role for the ATPase activity of MLH1 beyond the activation of the endonuclease functions of its MMR partner PMS2. A preference for switch junctions with longer microhomologies in Mlh1(GR/GR) mice suggests that through its ATPase activity, MLH1 also has an impact in DNA end processing, favoring canonical NHEJ downstream of the DSB. Collectively, our study shows that the ATPase domain of MLH1 is important to transmit the CSR signaling cascade both upstream and downstream of the generation of DSBs.

The use of Bcl-2 over-expression to stabilize hybridomas specific to the HERG potassium channel.

We encountered a high degree of clonal hybridoma loss in the course of generating antibodies specific for the hERG potassium channel. A protein that is crucial for controlling heart rhythm, is abundant in parts of the brain and is abnormally expressed in some tumors. Intracellular domains of the protein were used for immunogens and generated adequate antibody responses in mice. Subsequent hybridomas created using Ag8 myeloma fusion partner yielded clones that secreted specific antibody but none could be successfully maintained in culture. A variety of mechanisms, including polyploidy inherent to hybridoma development or production of cytotoxic antibodies, may be responsible for eventual loss of cell viability by mechanisms that may include apoptosis. When spleen cells were fused to the NSO myeloma cell line that stably over-expresses the anti-apoptotic protein Bcl-2, hybridoma clones were generated that remained viable in culture with high level of hERG-specific antibody production. When the parental NSO cell line not over-expressing Bcl-2 was used, no stable hybridomas were produced. Antibodies secreted by NSO-Bcl-2 hybridomas were specific for hERG and performed well in immunoblot, immunoprecipitation and immunofluorescence assays. This work demonstrates a feasible option when faced with antigens that seem to be associated with clonal instability in the process of generating monoclonal antibodies.