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Summary: Despite the near ubiquitous presence of Ig‐based antibodies in vertebrates, IgE is unique to mammals. How and why it emerged remains mysterious. IgE expression is greatly constrained compared to other IgH isotypes. While other IgH isotypes are relatively abundant, soluble IgE has a truncated half‐life, and IgE plasma cells are mostly short‐lived. Despite its rarity, IgE is consequential and can trigger life‐threatening anaphylaxis. IgE production reflects a dynamic steady state with IgG memory B cells feeding short‐lived IgE production. Emerging evidence suggests that IgE may also potentially be produced in longer‐lived plasma cells as well, perhaps as an aberrancy stemming from its evolutionary roots from an antibody isotype that likely functioned more like IgG. As a late derivative of an ancient systemic antibody system, the benefits of IgE in mammals likely stems from the antibody system's adaptive recognition and response capability. However, the tendency for massive, systemic, and long‐lived production, common to IgH isotypes like IgG, were likely not a good fit for IgE. The evolutionary derivation of IgE from an antibody system that for millions of years was good at antigen de‐sensitization to now functioning as a highly specialized antigen‐sensitization function required heavy restrictions on antibody production—insufficiency of which may contribute to allergic disease. [ABSTRACT FROM AUTHOR]

We describe two types of IGH::BCL2 breakpoints involving the 5′ region of BCL2 (5′ BCL2). One was ins(14;18)(q32;q21q21) observed in 2 follicular lymphoma (FL) cases, in which IGH was cleaved at 3' of IGHD and 5' of IGHJ and BCL2 was cleaved at 5' BCL2 and downstream regions, and a 281- or 201-kilobase pair fragment containing the BCL2 protein-coding sequences was invertedly inserted into IGH. In another type observed in 2 FL and 2 chronic lymphocytic leukemia (CLL) cases, breakage and reunion occurred within the switch region associated with IGHM (Sµ) and 5′ BCL2, creating IGH Sµ::5′ BCL2 fusion sequences on der(18)t(14;18)(q32;q21). The former is considered to be mediated by VDJ-recombination, while the latter by the class switch recombination process. There were no particular features in FL or CLL cases with IGH::5′ BCL2 breakpoints compared with those with t(14;18)(q32;q21)/IGH::BCL2 involving the 3′ breakpoint cluster regions. [ABSTRACT FROM AUTHOR]

This article highlights four recent studies that investigated thymic B cells in both mice and humans. These studies provide new insights into the biology of thymic B cells, including their activation, differentiation, and their role in promoting the negative selection of thymocytes and the generation of regulatory T cells. The studies found that thymic B cells play a nonredundant role in T-cell tolerance and prevention of autoimmune diseases in mice, and they also identified unique characteristics of thymic B cells, such as their activation during the neonatal period and their strong interferon signature. Additionally, the studies discovered the presence of antibody-secreting cells in the thymus that recognize dietary antigens, suggesting a potential role for thymic B cells in systemic tolerance to dietary antigens. Overall, these findings contribute to our understanding of thymic B-cell development and function and highlight their importance in T-cell tolerance. [Extracted from the article]

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3′ regulatory region (3′RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only. [ABSTRACT FROM AUTHOR]

In the setting of hematopoietic stem cell transplantation (HSCT), Rituximab (RTX) is used for the treatment and prevention of EBV-associated post-transplantation lymphoproliferative disease or autoimmune phenomena such as autoimmune hemolytic anemia (AIHA). Persistent hypogammaglobulinemia and immunoglobulin substitution dependence has been observed in several patients after RTX treatment despite the normalization of total B cell numbers. We aimed to study whether this is a B cell intrinsic phenomenon. We analyzed four patients with different primary diseases who were treated with myeloablative conditioning and matched unrelated donor HSCT who developed persistent hypogammaglobulinemia after receiving RTX treatment. They all received RTX early after HSCT to treat EBV infection or AIHA post-HSCT. All patients showed normalized total B cell numbers but absent to very low IgG positive memory B cells, and three lacked IgA positive memory B cells. All of the patients had full donor chimerism, and none had encountered graft-versus-host disease. Sorted peripheral blood naïve B cells from these patients, when stimulated with CD40L, IL21, IL10 and anti-IgM, demonstrated intact B cell differentiation including the formation of class-switched memory B cells and IgA and IgG production. Peripheral blood T cell numbers including CD4 follicular T-helper (Tfh) cells were all within the normal reference range. In conclusion, in these four HSCT patients, the persistent hypogammaglobulinemia observed after RTX cannot be attributed to an acquired intrinsic B cell problem nor to a reduction in Tfh cell numbers. [ABSTRACT FROM AUTHOR]

Mammalian 53BP1 and replication timing regulatory factor 1 (RIF1) protect DNA double-strand breaks (DSBs) against nucleolytic degradation through the recruitment of the REV7-SHLD1-SHLD2-SHLD3 (shieldin) and CTC1–STN1–TEN1 (CST) complexes. Mouse models deficient in downstream effectors of DSB protection have enabled the comparative assessment of defects in V(D)J recombination and class switch recombination (CSR). Regulation of DSB resection is crucial for CSR but dispensable for V(D)J recombination. The DSB protection function of mammalian 53BP1 does not explain the CSR defect severity associated with its deficiency. V(D)J recombination and CSR rely on the dynamic reconfiguration of Tcr / Ig loci that is contributed by cohesin-mediated loop extrusion. We propose that mammalian 53BP1 mediates pre- and post-break functions related to Tcr / Ig dynamics and DSB end-tethering, respectively, during the two recombination reactions. The dissection of the key molecular activities contributed by 53BP1 to antibody gene diversification exemplifies the importance of considering the repair of V(D)J and class switch recombination DNA double-strand breaks in their architectural genomic contexts. The investigation of the close interplay between Tcr / Ig loci dynamics and the repair of these programmed breaks can provide a deeper understanding of the molecular bases of humoral immune responses. The DNA double-strand break (DSB) repair factor 53BP1 has long been implicated in V(D)J and class switch recombination (CSR) of mammalian lymphocyte receptors. However, the dissection of the underlying molecular activities is hampered by a paucity of studies [V(D)J] and plurality of phenotypes (CSR) associated with 53BP1 deficiency. Here, we revisit the currently accepted roles of 53BP1 in antibody diversification in view of the recent identification of its downstream effectors in DSB protection and latest advances in genome architecture. We propose that, in addition to end protection, 53BP1-mediated end-tethering stabilization is essential for CSR. Furthermore, we support a pre-DSB role during V(D)J recombination. Our perspective underscores the importance of evaluating repair of DSBs in relation to their dynamic architectural contexts. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage is the initial event of antibody gene-diversification processes such as class switch recombination (CSR) and somatic hypermutation (SHM). We previously reported the requirement of an AID-dependent decrease of topoisomerase 1 (Top1) for efficient DNA cleavage, but the underlying molecular mechanism has remained elusive. This study focuses on HuR / ELAVL1 , a protein that binds to AU-rich elements in RNA. HuR -knockout (KO) CH12 cells derived from murine B lymphoma cells were found to have lower CSR and hypermutation efficiencies due to decreased AID-dependent DNA cleavage levels. The HuR -KO CH12 cells do not show impairment in cell cycles and Myc expression, which have been reported in HuR-reduced spleen B cells. Furthermore, drugs that scavenge reactive oxygen species (ROS) do not rescue the lower CSR in HuR -KO CH12 cells, meaning that ROS or decreased c-Myc protein amount is not the reason for the deficiencies of CSR and hypermutation in HuR -KO CH12 cells. We show that HuR binds to Top1 mRNA and that complete deletion of HuR abolishes AID-dependent repression of Top1 protein synthesis in CH12 cells. Additionally, reduction of CSR to IgG3 in HuR -KO cells is rescued by knockdown of Top1, indicating that elimination of the AID-dependent Top1 decrease is the cause of the inefficiency of DNA cleavage, CSR and hypermutation in HuR -KO cells. These results show that HuR is required for initiation of antibody diversification and acquired immunity through the regulation of AID-dependent DNA cleavage by repressing Top1 protein synthesis. [ABSTRACT FROM AUTHOR]

Purpose: Primary B cell defects manifesting as predominantly antibody deficiencies result from variable inborn errors of the B cell lineage and their development, including impairments in early bone marrow development, class switch recombination (CSR), or terminal B cell differentiation. In this study, we aimed to investigate autoimmunity in monogenic patients with B cell development and differentiation defects. Methods: Patients with known genetic defects in the B cell development and differentiation were recruited from the Iranian inborn errors of immunity registry. Results: A total of 393 patients with a known genetic defect in the B cell development and differentiation (257 males; 65.4%) with a median age of 12 (6–20) years were enrolled in this study. After categorizing patients, 109 patients had intrinsic B cell defects. More than half of the patients had defects in one of the ATM (85 patients), BTK (76 patients), LRBA (34 patients), and DOCK8 (33 patients) genes. Fifteen patients (3.8%) showed autoimmune complications as their first manifestation. During the course of the disease, autoimmunity was reported in 81 (20.6%) patients at a median age of 4 (2–7) years, among which 65 patients had mixed intrinsic and extrinsic and 16 had intrinsic B cell defects. The comparison between patients with the mentioned four main gene defects showed that the patient group with LRBA defect had a significantly higher frequency of autoimmunity compared to those with other gene defects. Based on the B cell defect stage, 13% of patients with early B cell defect, 17% of patients with CSR defect, and 40% of patients who had terminal B cell defect presented at least one type of autoimmunity. Conclusion: Our results demonstrated that gene mutations involved in human B cell terminal stage development mainly LRBA gene defect have the highest association with autoimmunity. [ABSTRACT FROM AUTHOR]

Mature B cells notably diversify immunoglobulin (Ig) production through class switch recombination (CSR), allowing the junction of distant "switch" (S) regions. CSR is initiated by activation-induced deaminase (AID), which targets cytosines adequately exposed within single-stranded DNA of transcribed targeted S regions, with a specific affinity for WRCY motifs. In mammals, G-rich sequences are additionally present in S regions, forming canonical G-quadruplexes (G4s) DNA structures, which favor CSR. Small molecules interacting with G4-DNA (G4 ligands), proved able to regulate CSR in B lymphocytes, either positively (such as for nucleoside diphosphate kinase isoforms) or negatively (such as for RHPS4). G4-DNA is also implicated in the control of transcription, and due to their impact on both CSR and transcriptional regulation, G4-rich sequences likely play a role in the natural history of B cell malignancies. Since G4-DNA stands at multiple locations in the genome, notably within oncogene promoters, it remains to be clarified how it can more specifically promote legitimate CSR in physiology, rather than pathogenic translocation. The specific regulatory role of G4 structures in transcribed DNA and/or in corresponding transcripts and recombination hereby appears as a major issue for understanding immune responses and lymphomagenesis. [ABSTRACT FROM AUTHOR]

Background: The involvement of allergen‐specific (s)IgE in local allergic rhinitis (LAR) has been debated. Here, we investigate the effect of nasal allergen challenge with Dermatophagoides pteronyssinus (NAC‐DP) in mucosal and peripheral B‐cell subpopulations in LAR patients. Methods: Nine LAR, 5 allergic rhinitis (AR), and 5 non‐atopic healthy control (HC) individuals were subjected to a 3‐day NAC‐DP protocol, and nasal biopsies and blood samples were collected before and after provocation. Nasal biopsies were used for immunohistochemistry and gene expression studies, whereas the frequency of lymphocyte subsets and basophil activation test (BAT) were analyzed in blood samples by flow cytometry. sIgG was measured in sera. Results: NAC‐DP induced an increase in IgE+CD38+ plasmablasts in the nasal mucosa of LAR patients, but not in AR or HC individuals. Markers of sequential recombination to IgE (εCSR) (from IgG) were observed in 33% of LAR, 20% of AR, and 0% of HC subjects. NAC‐DP increased the proportion of peripheral CD19+CD20+CD38+ plasmablasts in AR and LAR patients, but not in HC. Expression of the mucosal homing receptor CXCR3 in peripheral CD19+CD20+CD38+ plasmablasts from LAR, AR, and HC individuals was 7%, 5%, and 0.5%, respectively. In vitro DP stimulation increased proliferating CD19+CD20+CD38+ plasmablasts in LAR and AR patients, but not in HC. Serum DP‐sIgG was higher in LAR and AR patients as compared to HC. BAT was positive in 33%, 100%, and 0% of LAR, AR, and HC subjects, respectively. Conclusion: These results suggest that allergen exposure induces the sequential εCSR of IgG+CD19+CD20+CD38+ plasmablasts in the nasal mucosa of LAR patients. [ABSTRACT FROM AUTHOR]

Several primary immunodeficiencies are caused by defects in the general DNA repair machinery as exemplified by the T‐B‐ radiosensitive SCID condition owing to impaired resolution of programmed DNA double‐strand breaks introduced by RAG1/2 during V(D)J recombination. The genome instability generally associated with these conditions results in an increased propensity to develop malignancies requiring genotoxic‐based anti‐cancer treatments. Moreover, the extent of immune deficiency often calls for hematopoietic stem cell transplantation as a definitive treatment, also requiring genotoxic‐based conditioning regimen prior to transplantation. In both cases, the underlying general DNA repair defect may result in catastrophic iatrogenic consequences. It is, therefore, of paramount importance to assess the functionality of the DNA repair apparatus prior to any genotoxic treatment when the exact molecular cause of the disease is unknown. For this purpose, two simple assays can be used on patients derived peripheral blood lymphocytes: (1) the PROMIDISα biomarker, based on the next‐generation sequencing analysis of the TCRα repertoire, will highlight specific signatures of DNA repair deficiencies; (2) direct analysis of the sensitivity of peripheral lymphocytes to ionizing radiation will formally identify patients at risk to develop toxicity toward genotoxic‐based treatments. [ABSTRACT FROM AUTHOR]

In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders. Synopsis: Immunodeficiency‐associated activation‐induced deaminase (AID) variants lacking the C‐terminus behave as dominant‐negative in antibody diversification. This study reveals co‐condensation as a basis of this dominant‐negative effect, with mutant AID proteins forming condensates and trapping wild‐type AID from its substrates. The AID C‐terminus is required for its recruitment to genomic antibody switch regionsC‐terminal‐deleted AID protein forms condensates in vitro and in vivoAID condensation causes a faulty distribution of deaminase activityC‐terminal‐deleted AID co‐condensates with wild‐type protein, resulting in the dominant‐negative effect [ABSTRACT FROM AUTHOR]

B cells fulfill an important role in the adaptive immunity. Upon activation and immunoglobulin (IG) class switching, these cells function in the humoral immunity compartment as plasma cells. For clinical applications, it can be important to quantify (switched) B cells accurately in a variety of body fluids and tissues of benign, inflammatory and malignant origin. For decades, flow cytometry and immunohistochemistry (IHC) have been the preferred methods for quantification. Although these methods are widely used, both depend on the accessibility of B cell epitopes and therefore require intact (fixed) cells. Whenever samples are low in quantity and/or quality, accurate quantification can be difficult. By shifting the focus from epitopes to DNA markers, quantification of B cells remains achievable. During differentiation and maturation, B cells are subjected to programmed genetic recombination processes like VDJ rearrangements and class switch recombination (CSR), which result in deletion of specific sequences of the IGH locus. These cell type-specific DNA "scars" (loss of sequences) in IG genes can be exploited as B cell markers in digital PCR (dPCR) based quantification methods. Here, we describe a novel, specific and sensitive digital PCR-based method to quantify mature and switched B cells in DNA specimens of benign and (copy number unstable) malignant origin. We compared this novel way of B cell quantitation with flow cytometric and immunohistochemical methods. Through cross-validation with flow cytometric sorted B cell subpopulations, we gained quantitative insights into allelic involvement in different recombination processes in the IGH locus. Our newly developed method is accurate and independent of the cellular context, offering new possibilities for quantification, even for (limited) small samples like liquid biopsies. • DNA based B cell quantification. • Digital PCR. • Allelic involvement. • IGH@ recombination. [ABSTRACT FROM AUTHOR]

Proper regulation of B‐cell function is essential for effective humoral immunity and maintenance of immune tolerance. Here, we found that FBW7 (F‐box/WD40 repeat‐containing protein 7) is highly expressed in germinal centre B and B1 cells, and confirmed that it has an intrinsic role in maintaining homeostasis of mature B cells and B‐1 cells. FBW7 deletion led to an impairment of antibody response, and although germinal centre formation was not affected, antibody class‐switch recombination and affinity maturation processes were defective. Likewise, memory immune response was severely impaired. Moreover, FBW7 ablation ameliorated the pathogenesis of an autoimmune disease model, collagen‐induced arthritis, by reducing the production of anti‐collagen II autoantibodies. Taken together, these data suggest that FBW7 may be an attractive target for developing new therapeutics for the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]

• p53 affects somatic hypermutation by action in non-B cells. • p53 defects in germinal center B cells do not affect variable region hypermutation. • p53 defects during class switch recombinatoin alter the mutagenesis pattern. • p53 defects in lymphoma cells lead to increased somatic hypermutation. p53 plays a major role in genome maintenance. In addition to multiple p53 functions in the control of DNA repair, a regulation of DNA damage bypass via translesion synthesis has been implied in vitro. Somatic hypermutation of immunoglobulin genes for affinity maturation of antibody responses is based on aberrant translesion polymerase action and must be subject to stringent control to prevent genetic alterations and lymphomagenesis. When studying the role of p53 in somatic hypermutation in vivo , we found altered translesion polymerase-mediated A:T mutagenesis in mice lacking p53 in all organs, but notably not in mice with B cell-specific p53 inactivation, implying that p53 functions in non-B cells may alter mutagenesis in B cells. During class switch recombination, when p53 prevents formation of chromosomal translocations, we in addition detected a B cell-intrinsic role for p53 in altering G:C and A:T mutagenesis. Thus, p53 regulates translesion polymerase activity and shows differential activity during somatic hypermutation versus class switch recombination in vivo. Finally, p53 inhibition leads to increased somatic hypermutation in human B lymphoma cells. We conclude that loss of p53 function may promote genetic instability via multiple routes during antibody diversification in vivo. [ABSTRACT FROM AUTHOR]

Checkpoint signaling in the context of a functional DNA damage response is crucial for the prevention of oncogenic transformation of cells. Our immune system, though, takes the risk of attenuated checkpoint responses during immunoglobulin diversification. B cells undergo continuous DNA damage and error‐prone repair of their immunoglobulin genes during the process of somatic hypermutation. An accompanying attenuation of the DNA damage response via the ATR–Chk1 axis in B cells is believed to allow for a better DNA damage tolerance and for evasion of apoptosis, so as to ensure mutations to be passed on. We sought to determine whether the downregulation of Chk1 could also directly influence the process of hypermutation in vivo by altering the relative activity of error‐prone DNA repair pathways. We analyzed the humoral response and the hypermutation process in mice whose B cells express reduced levels of the Chk1 protein. We found that Chk1 heterozygosity limits the accumulation of mutations in the immunoglobulin loci, likely by impacting on the survival of B cells as they accumulate DNA damage. Nevertheless, we unveiled an unanticipated role for Chk1 downregulation in favoring A/T mutagenesis at the antibody‐variable regions during hypermutation. Even though immunoglobulin mutagenesis was found to be reduced, Chk1 signaling attenuation allows for sustained mutagenesis outside the immunoglobulin loci. Our study thus reveals that a proper Chk1 dosage is crucial for adequate somatic hypermutation in B cells. [ABSTRACT FROM AUTHOR]

Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery. [Display omitted] • H2AK5ac and H2AK9ac respond rapidly to DNA damage and are deacetylated by MiDAC • BRD4 reads the H2AK5acK9ac mark and phase separates with KU at DSB-proximal chromatin • BRD4-KU condensates block the assembly of NHEJ synaptic complex • MiDAC suppresses BRD4-KU molecular crowding by removal of di-acetyls from H2AK5K9 Bao et al. reveal that the histone deacetylase complex MiDAC promotes H2AK5K9 deacetylation and licenses NHEJ synaptic complex assembly. In the context of MiDAC ablation, elevated H2AK5acK9ac serves as a docking signal for BRD4 to compartmentalize with KU around damaged chromatin, and this aberrant condensation inhibits DNA end synapsis and reduces genome stability. [ABSTRACT FROM AUTHOR]

ATM is often dubbed the master regulator of the DNA double stranded break (DSB) response. Since proper induction and repair of DNA DSBs forms the core of immunological diversity, it is surprising that patients with ataxia telangiectasia generally have a mild immunodeficiency in contrast to other DSB repair syndromes. In this review, we address this discrepancy by delving into the functions of ATM in DSB repair and cell cycle control and translate these to adaptive immunity. We conclude that ATM, despite its myriad functions, is not an absolute requirement for acquiring sufficient levels of immunological diversity to prevent severe viral and opportunistic infections. There is, however, a more clinically pronounced antibody deficiency in ataxia telangiectasia due to disturbed class switch recombination. The functions of ATM are closely intertwined with the processes that generate immunological diversity. The ATM DNA double-stranded break (DSB) response triggers the formation of a network of recruited proteins (including the MRN complex, MDC1, 53BP1) participating in tethering the break ends together. ATM deficiency reduces DSB end tethering, coding end hairpin resolution, and repair, in V(D)J-recombination. This results in a lower success rate of V(D)J recombination, with reduced receptor diversity and increased risk of (oncogenic) translocations in ATM-deficient patients compared with healthy controls. ATM participates in a positive feedback loop in CSR, through phosphorylation of Activation Induced cytidine Deaminase (AID). The end tethering properties of ATM and 53BP1 seem to be more essential for CSR, compared with V(D)J-recombination. Clinically, ATM deficiency primarily causes antibody deficiency. [ABSTRACT FROM AUTHOR]

Immunoglobulin (Ig) class switch recombination (CSR) is the process occurring in mature B cells that diversifies the effector component of antibody responses. CSR is initiated by the activity of the B cell-specific enzyme activation-induced cytidine deaminase (AID), which leads to the formation of programmed DNA double-strand breaks (DSBs) at the Ig heavy chain (Igh) locus. Mature B cells use a multilayered and complex regulatory framework to ensure that AID-induced DNA breaks are channeled into productive repair reactions leading to CSR, and to avoid aberrant repair events causing lymphomagenic chromosomal translocations. Here, we review the DNA repair pathways acting on AID-induced DSBs and their functional interplay, with a particular focus on the latest developments in their molecular composition and mechanistic regulation. Mature B cells rely on a multilayered regulatory framework to ensure that S region DSBs are preferentially channeled into the NHEJ pathway. The structure of AID-induced breaks influences both the DSB end-processing mode and end-joining pathway choice. Both the SSA factor RAD52 and the A-EJ protein HMCES contribute strand pairing activities during repair of S region DSBs. Repair of AID-initiated breaks is influenced by Igh locus-specific organizational features that ensure productive end-joining events leading to CSR. 53BP1 contributes both structural and resection modulatory roles to the regulation of CSR repair outcomes. Shieldin and CST complexes are 53BP1 and Rif1 downstream effectors, and actively counteract the processing of S region DSBs into ssDNA by combining inhibition of DNA end resection and limited fill-in synthesis of resected tracks. [ABSTRACT FROM AUTHOR]

Summary: During the immune response, B cells can enter the memory pathway, which is characterized by class switch recombination (CSR), or they may undergo plasma cell differentiation (PCD) to secrete immunoglobulin. Both of these processes occur in activated B cells, which are reported to relate to membrane‐association proteins and adaptors. Protein 4.1R acts as an adaptor, linking membrane proteins to the cytoskeleton, and is involved in many cell events such as cell activation and differentiation, and cytokine secretion. However, the effect of 4.1R on regulating B‐cell fate is unclear. Here, we show an important association between B‐cell fate and 4.1R. In vitro, primary B cells were stimulated with lipopolysaccharide combined with interleukin‐4; results showed that 4.1R‐deficient (4.1R–/–) cells compared with wild‐type (4.1R+/+) B cells augmented expression of activation‐induced cytidine deaminase and germline, resulting in increased IgG1+ B cells, whereas the secretion of IgG1 and IgM was reduced, and CD138+ B cells were also decreased. Throughout the process, 4.1R regulated canonical nuclear factor (NF‐κB) rather than non‐canonical NF‐κB to promote the expression of CSR complex components, leading to up‐regulation of B‐cell CSR. In contrast, 4.1R‐deficient B cells showed reduced expression of Blimp‐1, which caused B cells to down‐regulate PCD. Furthermore, over‐activation of canonical NF‐κB may induce apoptosis signaling to cause PCD apoptosis to reduce PCD number. In summary, our results suggest that 4.1R acts as a B‐cell fate regulator by inhibiting the canonical NF‐κB signaling pathway. [ABSTRACT FROM AUTHOR]

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ~25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR. [ABSTRACT FROM AUTHOR]

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion–circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy. [ABSTRACT FROM AUTHOR]

Background: HIV-1 Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through modulation of several host cell functions. In addition to pro-apoptotic and cytostatic properties, Vpr can redirect cellular E3 ubiquitin ligases (such as DCAF1-Cul4A E3 ligase complex) to target many host proteins and interfere with their functions. Among them, Vpr binds the uracil DNA glycosylase UNG2, which controls genome uracilation, and induces its specific degradation leading to loss of uracil removal activity in infected cells. Considering the essential role of UNG2 in antibody diversification in B-cells, we evaluated the impact of Vpr on UNG2 fate in B lymphocytes and examined the functional consequences of UNG2 modulations on class switch recombination (CSR).Methods: The impact of Vpr-induced UNG2 deregulation on CSR proficiency was evaluated by using virus-like particles able to deliver Vpr protein to target cells including the murine model CSR B cell line CH12F3 and mouse primary B-cells. Co-culture experiments were used to re-examine the ability of Vpr to be released by HIV-1 infected cells and to effectively accumulate in bystander B-cells. Vpr-mediated UNG2 modulations were monitored by following UNG2 protein abundance and uracil removal enzymatic activity.Results: In this study we report the ability of Vpr to reduce immunoglobulin class switch recombination (CSR) in immortalized and primary mouse B-cells through the degradation of UNG2. We also emphasize that Vpr is released by producing cells and penetrates bystander B lymphocytes.Conclusions: This work therefore opens up new perspectives to study alterations of the B-cell response by using Vpr as a specific CSR blocking tool. Moreover, our results raise the question of whether extracellular HIV-1 Vpr detected in some patients may manipulate the antibody diversification process that engineers an adapted response against pathogenic intruders and thereby contribute to the intrinsic B-cell humoral defect reported in infected patients. [ABSTRACT FROM AUTHOR]

Summary: Immunoglobulins emerging from B lymphocytes and capable of recognizing almost all kinds of antigens owing to the extreme diversity of their antigen‐binding portions, known as variable (V) regions, play an important role in immune responses. The exons encoding the V regions are known as V (variable), D (diversity), or J (joining) genes. V, D, J segments exist as multiple copy arrays on the chromosome. The recombination of the V(D)J gene is the key mechanism to produce antibody diversity. The recombinational process, including randomly choosing a pair of V, D, J segments, introducing double‐strand breaks adjacent to each segment, deleting (or inverting in some cases) the intervening DNA and ligating the segments together, is defined as V(D)J recombination, which contributes to surprising immunoglobulin diversity in vertebrate immune systems. To enhance both the ability of immunoglobulins to recognize and bind to foreign antigens and the effector capacities of the expressed antibodies, naive B cells will undergo class switching recombination (CSR) and somatic hypermutation (SHM). However, the genetics mechanisms of V(D)J recombination, CSR and SHM are not clear. In this review, we summarize the major progress in mechanism studies of immunoglobulin V(D)J gene recombination and CSR as well as SHM, and their regulatory mechanisms. [ABSTRACT FROM AUTHOR]

One of the most profound enigmas in B cell biology is how activation-induced deaminase (AID) is targeted to a very small region of DNA in the immunoglobulin loci. Two specific regions are singled out: the variable region of 2 kb that contains rearranged genes on the heavy, κ light, and λ light chain loci, and the switch region of ∼4 kb that contains an extensive stretch of G:C rich DNA on the heavy chain locus. Transcription is required for AID recruitment; however, many genes are also highly transcribed and do not undergo the catastrophic mutagenesis that occurs in variable and switch regions. The DNA sequences of these regions cause RNA polymerase II to accumulate for an extended distance of 2–4 kb. The stalled polymerases then recruit the transcription cofactor Spt5, and AID, which deaminates cytosines to uracils in exposed transcription bubbles. Thus, the immunoglobulin loci are unique in that a favorable combination of DNA sequences and 3′ transcription enhancers make them the perfect storm for AID-induced somatic hypermutation. [ABSTRACT FROM AUTHOR]

Secretory immunoglobulin A (SIgA), the most abundant antibody isotype in the body, maintains a mutual relationship with commensal bacteria and acts as a primary barrier at the mucosal surface. Colonization by commensal bacteria induces an IgA response, at least partly through a T-cell-independent process. However, the mechanism underlying the commensal-bacteria-induced T-cell-independent IgA response has yet to be fully clarified. Here, we show that commensal-bacteria-derived butyrate promotes T-cell-independent IgA class switching recombination (CSR) in the mouse colon. Notably, the butyrate concentration in human stools correlated positively with the amount of IgA. Butyrate up-regulated the production of transforming growth factor β1 and all- trans retinoic acid by CD103+CD11b+ dendritic cells, both of which are critical for T-cell-independent IgA CSR. This effect was mediated by G-protein-coupled receptor 41 (GPR41/FFA3) and GPR109a/HCA2, and the inhibition of histone deacetylase. The butyrate-induced IgA response reinforced the colonic barrier function, preventing systemic bacterial dissemination under inflammatory conditions. These observations demonstrate that commensal-bacteria-derived butyrate contributes to the maintenance of the gut immune homeostasis by facilitating the T-cell-independent IgA response in the colon. [ABSTRACT FROM AUTHOR]

Background: Hyper-immunoglobulin M (HIGM) syndrome is a rare heterogeneous group of primary immunodeficiency disorders characterized by low or absent serum levels of IgG and IgA along with normal or elevated serum levels of IgM. Methods: Clinical and immunological data were collected from the 75 patients' medical records diagnosed in Children's Medical Center affiliated to Tehran University Medical Sciences and other Universities of Medical Sciences in Iran. Among 75 selected patients, 48 patients (64%) were analyzed genetically using targeted and whole-exome sequencing. Results: The ratio of male to female was 2.9:1. The median age at the onset of the disease, time of diagnosis, and diagnostic delay were 10.5, 50, and 24 months, respectively. Pneumonia and lower respiratory tract infections (61.3%) were the most common complications. Responsible genes were identified in 35 patients (72.9%) out 48 genetically analyzed patients. Cluster of differentiation 40 ligand gene was the most mutated gene observed in 24 patients (68.5%) followed by activation-induced cytidine deaminase gene in 7 patients, lipopolysaccharide-responsive and beige-like anchor (1 patient), nuclear factor-kappa-B essential modulator (1 patient), phosphoinositide-3-kinase regulatory subunit 1 (1 patient), and nuclear factor kappa B subunit 1 (1 patient) genes. Nineteen (25.3%) patients died during the study period, and pneumonia was the major cause of death occurred in 6 (31.6%) patients. Conclusion: Physicians in our country should carefully pay attention to respiratory tract infections and pneumonia, particularly in patients with a positive family history. Further investigations are required for detection of new genes and pathways resulting in HIGM phenotype. [ABSTRACT FROM AUTHOR]

Genetic variants in genes acting during the maturation process of immature B-cell to differentiated plasma cell could influence the risk of developing multiple myeloma (MM). During B-cell maturation, several programmed genetic rearrangements occur to increase the variation of the immunoglobulin chains. Class switch recombination (CSR) is one of the most important among these mechanisms. Germline polymorphisms altering even subtly this process could play a role in the etiology and outcome of MM. We performed an association study of 30 genetic variants in the key CSR genes, using 2632 MM patients and 2848 controls from the International Multiple Myeloma rESEarch (IMMEnSE) consortium, the Heidelberg MM Group and the ESTHER cohort. We found an association between LIG4-rs1555902 and decreased MM risk, which approached statistical significance, as well as significant associations between AICDA-rs3794318 and better outcome. Our results add to our knowledge on the genetic component of MM risk and survival. [ABSTRACT FROM AUTHOR]

Abstract Hyper Immunoglobulin M syndrome (HIGM) is a rare primary immunodeficiency disorder characterized by low or absent levels of serum IgG, IgA, IgE and normal or increased levels of serum IgM. Various X-linked and autosomal recessive/dominant mutations have been reported as the underlying cause of the disease. Based on the underlying genetic defect, the affected patients present a variety of clinical manifestations including pulmonary and gastrointestinal complications, autoimmune disorders, hematologic abnormalities, lymphoproliferation and malignancies which could be controlled by multiple relevant therapeutic approaches. Herein, the epidemiology, pathogenesis, clinical manifestations, diagnosis, management, prognosis and treatment in patients with HIGM syndrome have been reviewed. Highlights • Various mutations in B cell pathway • The presence of heterogeneous manifestations in HIgM patients • Therapeutic approaches among HIGM patients [ABSTRACT FROM AUTHOR]

An essential step in the development of effective antiviral humoral responses is cytokine-triggered class switch recombination resulting in the production of antibodies of a specific isotype. Most viral and parasitic infections in mice induce predominantly IgG2a-specific antibody responses that are stimulated by interferon gamma (IFN-γ). However, in some mice deficient in IFN-γ, class switching to IgG2a antibodies is relatively unaffected, indicating that another signal(s) can be generated upon viral or parasitic infections that trigger this response. Here, we found that a single recessive locus, provisionally called IFN-γ-independent IgG2a (Igii), confers the ability to produce IFN-γ-independent production of IgG2a antibodies upon retroviral infection. The Igii locus was mapped to chromosome 9 and was found to function in the radiation-resistant compartment. Thus, our data implicate nonhematopoietic cells in activation of antiviral antibody responses in the absence of IFN-γ. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greater degree of predictability than is achievable with conventional induction of CSR. Here we show that Cas9-mediated nicks separated by up to 250 nucleotides on opposite strands can mediate CSR. Staggered double-stranded breaks (DSBs) result in more end resection and junctional microhomology than blunt DSBs. Moreover, Myc-Igh chromosomal translocations, which are carried out primarily by alternative end joining (A-EJ), were preferentially induced by 5' DSBs. These data indicate that DSBs with 5' overhangs skew intrachromosomal and interchromosomal end-joining toward A-EJ. In addition to lending potential insight to AID-mediated phenomena, this work has broader carryover implications in DNA repair and lymphomagenesis. [ABSTRACT FROM AUTHOR]

Ig heavy chain (IgH) class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double-strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical nonhomologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining that is more biased to use longer junctional microhomologies (MHs). Deficiency for DSB response (DSBR) factors, including ataxia telangiectasia-mutated (ATM) and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B-lymphoma cells. Compared with wild-type, CSR and c-myc to S region translocation junctions in the absence of 53BP1, and, to a lesser extent, other DSBR factors, have increased MH utilization; indeed, 53BP1-deficient MH profiles resemble those associated with C-NHEJ deficiency. However, translocation junctions between c-myc DSB and general DSBs genome-wide are not MH-biased in ATMdeficient versus wild-type CH12F3 cells and are less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MHbiased end-joining in the absence of DSBR factors. [ABSTRACT FROM AUTHOR]

Activation–induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. [ABSTRACT FROM AUTHOR]

Bloom's syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene. This gene codes for BLM protein, which is a helicase involved in DNA repair. DNA repair is especially important for the development and maturation of the T and B cells. Since BLM is involved in DNA repair, we aimed to study if BLM deficiency affects T and B cell development and especially somatic hypermutation (SHM) and class switch recombination (CSR) processes. Clinical data of six BS patients was collected, and immunoglobulin serum levels were measured at different time points. In addition, we performed immune phenotyping of the B and T cells and analyzed the SHM and CSR in detail by analyzing IGHA and IGHG transcripts using next-generation sequencing. The serum immunoglobulin levels were relatively low, and patients had an increased number of infections. The absolute number of T, B, and NK cells were low but still in the normal range. Remarkably, all BS patients studied had a high percentage (20-80%) of CD4+ and CD8+ effector memory T cells. The process of SHM seems normal; however, the Ig subclass distribution was not normal, since the BS patients had more IGHG1 and IGHG3 transcripts. In conclusion, BS patients have low number of lymphocytes, but the immunodeficiency seems relatively mild since they have no severe or opportunistic infections. Most changes in the B cell development were seen in the CSR process; however, further studies are necessary to elucidate the exact role of BLM in CSR. [ABSTRACT FROM AUTHOR]

In B cells, Ig class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), the activity of which leads to DNA double-strand breaks (DSBs) within IgH switch (S) regions. Preferential targeting of AID-mediated DSBs to S sequences is critical for allowing diversification of antibody functions, while minimizing potential off-target oncogenic events. Here, we used gene targeted inactivation of histone methyltransferase (HMT) multiple myeloma SET domain (MMSET) in mouse B cells and the CH12F3 cell line to explore its role in CSR. We find that deletion of MMSET-II, the isoform containing the catalytic SET domain, inhibits CSR without affecting either IgH germline transcription or joining of DSBs within S regions by classical nonhomologous end joining (C-NHEJ). Instead, we find that MMSET-II inactivation leads to decreased AID recruitment and DSBs at the upstream donor Sµ region. Our findings suggest a role for the HMT MMSET in promoting AID-mediated DNA breaks during CSR. [ABSTRACT FROM AUTHOR]

Background Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/−). Objectives As maturation history of B-cell is “imprinted” during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes ( IGHV, IGLV, IGKV ), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin. Methods Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model. Results A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch μ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression. Conclusion These data suggest that cell-of origin is germinal center experienced and superantigen driven selected B-cell, in a stage between germinal center B-cell and plasma cell. [ABSTRACT FROM AUTHOR]

R-loops, three-strand structures consisting of mRNA hybridized to the complementary DNA and a single-stranded DNA loop, are formed in switch regions on the heavy-chain immunoglobulin locus. To determine if R-loops have a direct effect on any of the steps involved in isotype switching, we generated a transgenic mouse that over-expressed RNase H1, an enzyme that cleaves the RNA of RNA/DNA hybrids in B cells. R-loops in the switch μ region were depleted by 70% in ex vivo activated splenic B cells. Frequencies of isotype switching to IgG1, IgG2b, IgG2c, and IgG3 were the same as C57BL/6 control cells. However, somatic hypermutation was increased specifically on the transcribed strand from μ–γ joins, indicating that R-loops limit activation-induced (cytosine) deaminase access to the transcribed DNA strand. Our data suggest that, in the normal G + C-rich context of mammalian class switch recombination regions, R-loops are obligatory intermediates. Processing of the R-loops is needed to remove RNA allowing activation-induced (cytosine) deaminase to promote somatic hypermutation on both DNA strands to generate double-strand DNA breaks for efficient class switch recombination. One of the two cellular RNases H may assist in this process. [ABSTRACT FROM AUTHOR]

The eukaryotic RNA exosome is a well-conserved protein complex with ribonuclease activity implicated in RNA metabolism. Various families of non-coding RNAs have been identified as substrates of the complex, underscoring its role as a non-coding RNA processing/degradation unit. However, the role of RNA exosome and its RNA processing activity on DNA mutagenesis/alteration events have not been investigated until recently. B lymphocytes use two DNA alteration mechanisms, class switch recombination (CSR) and somatic hypermutation (SHM), to re-engineer their antibody gene expressing loci until a tailored antibody gene for a specific antigen is satisfactorily generated. CSR and SHM require the essential activity of the DNA activation-induced cytidine deaminase (AID). Causing collateral damage to the B-cell genome during CSR and SHM, AID induces unwanted (and sometimes oncogenic) mutations at numerous non-immunoglobulin gene sequences. Recent studies have revealed that AID's DNA mutator activity is regulated by the RNA exosome complex, thus providing an example of a mechanism that relates DNA mutagenesis to RNA processing. Here, we review the emergent functions of RNA exosome during CSR, SHM, and other chromosomal alterations in B cells, and discuss implications relevant to mechanisms that maintain B-cell genomic integrity. [ABSTRACT FROM AUTHOR]

Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 nuclease (CRISPR/Cas9) and Transcription Activator-Like Effector Nucleases (TALENs) are versatile tools for genome editing. Here we report a method to increase the frequency of Cas9-targeted cellular clones. Our method is based on a chimeric construct with a Blasticidin S Resistance gene ( bsr) placed out-of-frame by a surrogate target sequence. End joining of the CRISPR/Cas9-induced double-strand break on the surrogate target can place the bsr in frame, thus providing temporary resistance to Blasticidin S: this is used to enrich for cells where Cas9 is active. By this approach, in a real experimental setting, we disrupted the Aicda gene in ~70% of clones from CH12F3 lymphoma cells (>40% biallelically). With the same approach we knocked in a single nucleotide to reconstruct the frame of Aicda in these null cells, restoring the function in ~37% of the clones (less than 10% by the standard approach). Targeting of single nucleotide changes in other genes yielded analogous results. These results support our enrichment method as an efficient tool in genome editing. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with no known cure that affects at least five million people worldwide. Monozygotic twin concordance and familial aggregation studies strongly suggest that lupus results from genetic predisposition along with environmental exposures including UV light. The majority of the common risk alleles associated with genetic predisposition to SLE map to genes associated with the immune system. However, evidence is emerging that implicates a role for aberrant DNA repair in the development of lupus. Here we summarize our current knowledge of the potential association of lupus with mutations in DNA repair genes. We also discuss how defective or aberrant DNA repair could lead to the development of lupus. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs. [ABSTRACT FROM AUTHOR]

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3' regulatory region (3'RR) in a stimulus-dependent manner. Why the 3'RR-mediated up- regulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3'RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3'RR in developing B cells. [ABSTRACT FROM AUTHOR]

Autoantibodies of the IgG subclass are pathogenic in a number of autoimmune disorders such as systemic lupus erythomatosus. The presence of circulating IgE autoantibodies in autoimmune patients has also been known for almost 40 years. Despite their role in allergies, IgE autoantibodies are not associated with a higher rate of atopy in these patients. However, recently they have been recognized as active drivers of autoimmunity through mechanisms involving the secretion of Type I interferons by plasmacytoid dendritic cells (pDC), the recruitment of basophils to lymph nodes, and the activation of adaptive immune responses through B and T cells. Here, we will review the formation, prevalence, affinity, and roles of the IgE autoantibodies that have been described in autoimmunity. We also present novel evidence supporting that triggering of IgE receptors in pDC induces LC3-associated phagocytosis, a cellular process also known as LAP that is associated with interferon responses. The activation of pDC with immune complexes formed by DNA-specific IgE antibodies also induce potent B-cell differentiation and plasma cell formation, which further define IgE’s role in autoimmune humoral responses. [ABSTRACT FROM PUBLISHER]

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we propose that 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability. [Display omitted] • SLFN5 regulates the chromatin topology of 53BP1 at DSB sites • SLFN5 links damaged chromatin mobility to 53BP1 higher-order topology • SLFN5's ATPase is required in the 53BP1 topological rearrangement • Loss of SLFN5 impairs NHEJ and causes genomic instability Huang et al. show that SLFN5 promotes the dynamic behavior of DSBs and induces a 53BP1 higher-order topological arrangement to promote non-homologous end joining, end protection, and genome stability. [ABSTRACT FROM AUTHOR]

Interactions between transcription and cohesin-mediated loop extrusion can influence 3D chromatin architecture. However, their relevance in biology is unclear. Here, we report a direct role for such interactions in the mechanism of antibody class switch recombination (CSR) at the murine immunoglobulin heavy chain locus (Igh). Using Tri-C to measure higher-order multiway interactions on single alleles, we find that the juxtaposition (synapsis) of transcriptionally active donor and acceptor Igh switch (S) sequences, an essential step in CSR, occurs via the interaction of loop extrusion complexes with a de novo topologically associating domain (TAD) boundary formed via transcriptional activity across S regions. Surprisingly, synapsis occurs predominantly in proximity to the 3′ CTCF-binding element (3′CBE) rather than the Igh super-enhancer, suggesting a two-step mechanism whereby transcription of S regions is not topologically coupled to synapsis, as has been previously proposed. Altogether, these insights advance our understanding of how 3D chromatin architecture regulates CSR. [Display omitted] • The multiway interactome of single Igh alleles undergoing CSR is revealed by Tri-C • Transcription of switch (S) regions is topologically uncoupled from S-S synapsis • Transcription creates a de novo TAD boundary in Igh S regions • Interaction of cohesin complexes with TAD boundaries underpins S-S synapsis CSR generates new antibody isotypes via deletional recombination between transcribed and juxtaposed (synapsed) switch (S) sequences of the immunoglobulin heavy chain genes. Here, the authors report that transcription creates a topological barrier that facilitates S-S synapsis, thereby revealing how interactions between cohesin and transcription complexes directly influence the mechanism of a major biological process. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans -acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding. [ABSTRACT FROM AUTHOR]

Activation induced cytidine deaminase (AID) is an essential enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) during secondary immune response. Mutations in the AICDA gene are responsible for Hyper IgM 2 syndrome where both CSR and SHM or only CSR are affected. Indeed, triggering either of the two mechanisms requires the DNA deamination activity of AID. Besides, different domains of AID may be differentially involved in CSR and SHM through their interaction with specific cofactors. Herein, we studied the AID-induced SHM activity of the AID-His130Pro mutant identified in a patient with Hyper IgM 2 syndrome. AID mutagenic activity was monitored by the reversion of nonsense mutations of the EGFP gene assessed by flow cytometry. We found that the His130Pro mutation, which affects CSR, preserves AID mutagenic activity. Indeed, the His130 residue is located in a putative specific CSR region in the APOBEC-like domain, known to involve CSR specific cofactors that probably play a major role in AID physiological activities. [ABSTRACT FROM AUTHOR]

Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the J H cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3′ regulatory region (3′RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease. [ABSTRACT FROM AUTHOR]

Nonhomologous end-joining (NHEJ) is the major DNA doublestrand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigenstimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4-/- cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR. [ABSTRACT FROM AUTHOR]

The enzyme activation-induced deaminase (AID) targets the immunoglobulin loci in activated B cells and creates DNA mutations in the antigen-binding variable region and DNA breaks in the switch region through processes known, respectively, as somatic hypermutation and class switch recombination. AID deaminates cytosine to uracil in DNA to create a U:G mismatch. During somatic hypermutation, the MutSα complex binds to the mismatch, and the error-prone DNA polymerase η generates mutations at A and T bases. During class switch recombination, both MutSα and MutLα complexes bind to the mismatch, resulting in double-strand break formation and end-joining. This review is centered on the mechanisms of how the MMR pathway is commandeered by B cells to generate antibody diversity. [ABSTRACT FROM AUTHOR]