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Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on short read sequencing data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n = 2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs and short read data for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions.

The adaptive immune response in vertebrates depends on the expression of antigen-specific receptors in lymphocytes. T-cell receptor (TCR) gene expression is exquisitely regulated during thymocyte development to drive the generation of αβ and γδ T lymphocytes. The TCRα, TCRβ, TCRγ, and TCRδ genes exist in two different configurations, unrearranged and rearranged. A correctly rearranged configuration is required for expression of a functional TCR chain. TCRs can take the form of one of three possible heterodimers, pre-TCR, TCRαβ, or TCRγδ which drive thymocyte maturation into αβ or γδ T lymphocytes. To pass from an unrearranged to a rearranged configuration, global and local three dimensional (3D) chromatin changes must occur during thymocyte development to regulate gene segment accessibility for V(D)J recombination. During this process, enhancers play a critical role by modifying the chromatin conformation and triggering noncoding germline transcription that promotes the recruitment of the recombination machinery. The different signaling that thymocytes receive during their development controls enhancer activity. Here, we summarize the dynamics of long-distance interactions established through chromatin regulatory elements that drive transcription and V(D)J recombination and how different signaling pathways are orchestrated to regulate the activity of enhancers to precisely control TCR gene expression during T-cell maturation.

Lymphocytes are endowed with unique and specialized enzymatic mutagenic properties that allow them to diversify their antigen receptors, which are crucial sensors for pathogens and mediators of adaptive immunity. During lymphocyte development, the antigen receptors expressed by B and T lymphocytes are assembled in an antigen-independent fashion by ordered variable gene segment recombinations (V(D)J recombination), which is a highly ordered and regulated process that requires the recombination activating gene products 1 & 2 (RAG1, RAG2). Upon activation by antigen, B lymphocytes undergo additional diversifications of their immunoglobulin B-cell receptors. Enzymatically induced somatic hypermutation (SHM) and immunoglobulin class switch recombination (CSR) improves the affinity for antigen and shape the effector function of the humoral immune response, respectively. The activation-induced cytidine deaminase (AID) enzyme is crucial for both SHM and CSR. These processes have evolved to both utilize as well as evade different DNA repair and DNA damage response pathways. The delicate balance between enzymatic mutagenesis and DNA repair is crucial for effective immune responses and the maintenance of genomic integrity. Not surprisingly, disturbances in this balance are at the basis of lymphoid malignancies by provoking the formation of oncogenic mutations and chromosomal aberrations. In this review, we discuss recent mechanistic insight into the regulation of RAG1/2 and AID expression and activity in lymphocytes and the complex interplay between these mutagenic enzymes and DNA repair and DNA damage response pathways, focusing on the base excision repair and mismatch repair pathways. We discuss how disturbances of this interplay induce genomic instability and contribute to oncogenesis.

To be effective, the adaptive immune response requires a large repertoire of antigen receptors, which are generated through V(D)J recombination in lymphoid precursors. These precursors must be protected from DNA damage-induced cell death, however, because V(D)J recombination generates double-strand breaks and may activate p53. Here we show that the BTB/POZ domain protein Miz-1 restricts p53-dependent induction of apoptosis in both pro-B and DN3a pre-T cells that actively rearrange antigen receptor genes. Miz-1 exerts this function by directly activating the gene for ribosomal protein L22 (Rpl22), which binds to p53 mRNA and negatively regulates its translation. This mechanism limits p53 expression levels and thus contains its apoptosis-inducing functions in lymphocytes, precisely at differentiation stages in which V(D)J recombination occurs. Significance V(D)J recombination occurs in lymphoid precursors to enable their maturation, but also induces DNA damage. Thus, it has been proposed that the activity of the tumor suppressor and gatekeeper protein p53 must be controlled during this process to prevent premature induction of apoptosis. In this study, we show that the transcription factor Miz-1 can exert such a function. Miz-1 activates expression of the ribosomal protein Rpl22, which in turn controls the translation of p53 specifically in lymphoid precursors. We propose that this Miz-1–Rpl22–p53 pathway prevents p53 from inducing cell death as a response to V(D)J recombination in lymphoid precursors from both the T-lineage and the B-lineage.

In this Review, I present evidence supporting a multifactorial causation of childhood acute lymphoblastic leukaemia (ALL), a major subtype of paediatric cancer. ALL evolves in two discrete steps. First, in utero initiation by fusion gene formation or hyperdiploidy generates a covert, pre-leukaemic clone. Second, in a small fraction of these cases, the postnatal acquisition of secondary genetic changes (primarily V(D)J recombination-activating protein (RAG) and activation-induced cytidine deaminase (AID)-driven copy number alterations in the case of ETS translocation variant 6 (ETV6)–runt-related transcription factor 1 (RUNX1)+ ALL) drives conversion to overt leukaemia. Epidemiological and modelling studies endorse a dual role for common infections. Microbial exposures earlier in life are protective but, in their absence, later infections trigger the critical secondary mutations. Risk is further modified by inherited genetics, chance and, probably, diet. Childhood ALL can be viewed as a paradoxical consequence of progress in modern societies, where behavioural changes have restrained early microbial exposure. This engenders an evolutionary mismatch between historical adaptations of the immune system and contemporary lifestyles. Childhood ALL may be a preventable cancer.

53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1 ) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination 1 , 2 , and reduced fidelity of long-range V(D)J recombination 3 . The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres 4 , and its unrestricted activity in Brca1- deficient cellular and tumour models causes genomic instability and oncogenesis 5 – 7 . Cells that lack core non-homologous end joining proteins are profoundly radiosensitive 8 , unlike 53BP1-deficient cells 9 , 10 , which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin—a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)—as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1- deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway. The specificity of 53BP1 and its co-factors for particular DNA substrates during non-homologous end joining (NHEJ) derives from REV7–shieldin, a four-subunit DNA-binding complex that is required for REV7-dependent NHEJ but not for REV7-dependent DNA interstrand cross-link repair.

The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to J H segments, before joining upstream V H segments to DJ H intermediates 1 . In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain 2 at the 3′ end of Igh contains an internal subdomain that spans the 5′ CBE anchor (IGCR1) 3 , the D H segments, and a RAG-bound recombination centre (RC) 4 . The RC comprises the J H -proximal D segment (DQ52), four J H segments, and the intronic enhancer (iEμ) 5 . Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS) 6 . D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented J H -23RSSs and V H -23RSSs, respectively 6 . Despite 12/23 compatibility, inversional D-to-J H joining via upstream D-12RSSs is rare 7 , 8 . Plasmid-based assays have attributed the lack of inversional D-to-J H joining to sequence-based preference for downstream D-12RSSs 9 , as opposed to putative linear scanning mechanisms 10 , 11 . As RAG linearly scans convergent CBE-anchored chromatin loops 4 , 12 – 14 , potentially formed by cohesin-mediated loop extrusion 15 – 18 , we revisited its scanning role. Here we show that the chromosomal orientation of J H -23RSS programs RC-bound RAG to linearly scan upstream chromatin in the 3′ Igh subdomain for convergently oriented D-12RSSs and, thereby, to mediate deletional joining of all D segments except RC-based DQ52, which joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of J H segments, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3′ Igh subdomain, in which scanning can be impeded by targeted binding of nuclease-dead Cas9, by transcription through repetitive Igh switch sequences, and by the 3′ Igh CBE-based loop anchor. Each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High-resolution mapping of chromatin interactions in the RC reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. V(D)J recombination in B cells involves cohesin-mediated extrusion of chromatin loops to present DNA targets for cleavage and joining.

Key Points Recombination-activating gene (RAG) mutations in humans are associated with a broad spectrum of clinical phenotypes, ranging from severe, early-onset infections to inflammation and autoimmunity. There is a correlation between the severity of the clinical and immunological phenotypes and the recombination activity of the mutant RAG protein, and hypomorphic mutations that severely affect recombination activity are associated with restriction of the T cell and B cell repertoires. However, environmental factors may also contribute to determining the disease phenotype. Crystal structure and cryo-electron microscopy studies have revealed the structure of the heterotetrameric RAG complex bound to DNA. Fine definition of this structure has also offered important insights into the disease-causing effects of naturally occurring RAG mutations. Studies in patients and in mice have demonstrated that RAG mutations affect central and peripheral T cell and B cell tolerance, including defective expression of autoimmune regulator (AIRE), reduced number and function of regulatory T cells, impaired receptor editing and increased levels of B cell-activating factor (BAFF), allowing the rescue of self-reactive B cells. A broad range of autoantibodies has been demonstrated in patients with RAG mutations presenting with inflammation and autoimmunity. Neutralizing antibodies specific for interferon-α (IFNα) and IFNω have been documented particularly in patients with a history of severe viral infections. Recent data indicate that RAG expression during the early stages of lymphoid development selects cells with improved fitness. NK cells from Rag −/− mice have an activated phenotype and increased cytotoxicity. If confirmed in humans, these data may account for the high rate of graft rejection observed after unconditioned haematopoietic stem cell transplantation in patients with RAG deficiency. The wide diversity of clinical and immunological phenotypes of patients with RAG deficiency, combined with structural characterization of the RAG protein complex, have provided new mechanistic insights into RAG protein function. The recombination-activating gene 1 (RAG1) and RAG2 proteins initiate the V(D)J recombination process, which ultimately enables the generation of T cells and B cells with a diversified repertoire of antigen-specific receptors. Mutations of the RAG genes in humans are associated with a broad spectrum of clinical phenotypes, ranging from severe combined immunodeficiency to autoimmunity. Recently, novel insights into the phenotypic diversity of this disease have been provided by resolving the crystal structure of the RAG complex, by developing novel assays to test recombination activity of the mutant RAG proteins and by characterizing the molecular and cellular basis of immune dysregulation in patients with RAG deficiency.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

The antigen-binding variable regions of the B cell receptor (BCR) and of antibodies are encoded by exons that are assembled in developing B cells by V(D)J recombination 1 . The BCR repertoires of primary B cells are vast owing to mechanisms that create diversity at the junctions of V(D)J gene segments that contribute to complementarity-determining region 3 (CDR3), the region that binds antigen 1 . Primary B cells undergo antigen-driven BCR affinity maturation through somatic hypermutation and cellular selection in germinal centres (GCs) 2 , 3 . Although most GCs are transient 3 , those in intestinal Peyer’s patches (PPs)—which depend on the gut microbiota—are chronic 4 , and little is known about their BCR repertoires or patterns of somatic hypermutation. Here, using a high-throughput assay that analyses both V(D)J segment usage and somatic hypermutation profiles, we elucidate physiological BCR repertoires in mouse PP GCs. PP GCs from different mice expand public BCR clonotypes (clonotypes that are shared between many mice) that often have canonical CDR3s in the immunoglobulin heavy chain that, owing to junctional biases during V(D)J recombination, appear much more frequently than predicted in naive B cell repertoires. Some public clonotypes are dependent on the gut microbiota and encode antibodies that are reactive to bacterial glycans, whereas others are independent of gut bacteria. Transfer of faeces from specific-pathogen-free mice to germ-free mice restored germ-dependent clonotypes, directly implicating BCR selection. We identified somatic hypermutations that were recurrently selected in such public clonotypes, indicating that affinity maturation occurs in mouse PP GCs under homeostatic conditions. Thus, persistent gut antigens select recurrent BCR clonotypes to seed chronic PP GC responses. An analysis of the immunoglobulin repertoire of B cells in Peyer’s patch germinal centres in mice provides evidence for the selection of B cell receptor clonotypes by gut antigens and antigen-driven affinity maturation.