Activation-induced Deaminase Research Articles

Elucidation of the enigmatic IgD class-switch recombination via germline deletion of the IgH 3' regulatory region.

Classical class-switch recombination (cCSR) substitutes the Cμ gene with Cγ, Cε, or Cα, thereby generating IgG, IgE, or IgA classes, respectively. This activation-induced deaminase (AID)-driven process is controlled by the IgH 3' regulatory region (3'RR). Regulation of rare IgD CSR events has been enigmatic. We show that μδCSR occurs in mouse mesenteric lymph node (MLN) B cells and is AID-dependent. AID attacks differ from those in cCSR because they are not accompanied by extensive somatic hypermutation (SHM) of targeted regions and because repaired junctions exhibit features of the alternative end-joining (A-EJ) pathway. In contrast to cCSR and SHM, μδCSR is 3'RR-independent, as its absence affects neither breakpoint locations in Sμ- and Sδ-like (σ(δ)) nor mutation patterns at Sμ-σ(δ) junctions. Although mutations occur in the immediate proximity of the μδ junctions, SHM is absent distal to the junctions within both Sμ and rearranged VDJ regions. In conclusion, μδCSR is active in MLNs, occurs independently of 3'RR-driven assembly, and is even dramatically increased in 3'RR-deficient mice, further showing that its regulation differs from cCSR.

Humoral Immunity and CD4+ Th1 Cells Are Both Necessary for a Fully Protective Immune Response upon Secondary Infection with Brucella melitensis

Brucella spp are intracellular bacteria that cause brucellosis, one of the most common zoonoses in the world. Given the serious medical consequences of this disease, a safe and effective human vaccine is urgently needed. Efforts to develop this vaccine have been hampered by our lack of understanding of what constitutes a protective memory response against Brucella. In this study, we characterize the cells and signaling pathways implicated in the generation of a protective immune memory response following priming by the injection of heat-killed or live Brucella melitensis 16M. Using a panel of gene-deficient mice, we demonstrated that during a secondary recall response, both the Brucella-specific humoral response and CD4+ Th1 cells must act together to confer protective immunity in the spleen to B. melitensis infection. Humoral protective immunity is induced by the inoculation of both heat-killed and live bacteria, and its development does not require T cells, MyD88/IL-12p35 signaling pathways, or an activation-induced deaminase-mediated isotype switch. In striking contrast, the presence of memory IFN-γ-producing CD4+ Th1 cells requires the administration of live bacteria and functional MyD88/IL-12p35 pathways. In summary, our work identifies several immune markers closely associated with protective immune memory and could help to define a rational strategy to obtain an effective human vaccine against brucellosis.

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1tg C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5′-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy.

AIDing Chromatin and Transcription-Coupled Orchestration of Immunoglobulin Class-Switch Recombination.

Secondary diversification of the antibody repertoire upon antigenic challenge, in the form of immunoglobulin heavy chain (IgH) class-switch recombination (CSR) endows mature, naïve B cells in peripheral lymphoid organs with a limitless ability to mount an optimal humoral immune response, thus expediting pathogen elimination. CSR replaces the default constant (CH) region exons (Cμ) of IgH with any of the downstream CH exons (Cγ, Cε, or Cα), thereby altering effector functions of the antibody molecule. This process depends on, and is orchestrated by, activation-induced deaminase (AID), a DNA cytidine deaminase that acts on single-stranded DNA exposed during transcription of switch (S) region sequences at the IgH locus. DNA lesions thus generated are processed by components of several general DNA repair pathways to drive CSR. Given that AID can instigate DNA lesions and genomic instability, stringent checks are imposed that constrain and restrict its mutagenic potential. In this review, we will discuss how AID expression and substrate specificity and activity is rigorously enforced at the transcriptional, post-transcriptional, post-translational, and epigenetic levels, and how the DNA-damage response is choreographed with precision to permit targeted activity while limiting bystander catastrophe.

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR.

Mechanisms of oncogenic chromosomal translocations

Chromosome translocations are caused by inappropriate religation of two DNA double-strand breaks (DSBs) in heterologous chromosomes. These DSBs can be generated by endogenous or exogenous sources. Endogenous sources of DSBs leading to translocations include inappropriate recombination activating gene (RAG) or activation-induced deaminase (AID) activity during immune receptor maturation. Endogenous DSBs can also occur at noncanonical DNA structures or at collapsed replication forks. Exogenous sources of DSBs leading to translocations include ionizing radiation (IR) and cancer chemotherapy. Spatial proximity of the heterologous chromosomes is also important for translocations. While three distinct pathways for DNA DSB repair exist, mounting evidence supports alternative nonhomologous end joining (aNHEJ) as the predominant pathway through which the majority of translocations occur. Initiated by poly (ADP-ribose) polymerase 1 (PARP1), aNHEJ is utilized less frequently in DNA DSB repair than other forms of DSB repair. We recently found that PARP1 is essential for chromosomal translocations to occur and that small molecule PARP1 inhibitors, already in clinical use, can inhibit translocations generated by IR or topoisomerase II inhibition. These data confirm the central role of PARP1 in aNHEJ-mediated chromosomal translocations and raise the possibility of using clinically available PARP1 inhibitors in patients who are at high risk for secondary oncogenic chromosomal translocations.

Evaluation of a Novel Missense Activation-Induced Deaminase AID Mutation in a Child with Hyper IgM Syndrome: Is it a Pathogenic Mutation?

Hyper IgM Syndrome (HIGM) is a primary immunodeficiency associated with defects of CD40, CD40L, AID and UNG genes. In the case of a novel mutation, genetic testing requires secondary confirmation such as functional studies to establish a link between the clinical phenotype and the mutation identified.

Surfactant Protein A (SP-A) Reduces Human Rhinovirus 16 (RV16)-Induced Inflammatory Responses In Bronchial Epithelial Cells and Inhibits Viral Replication In H1-HeLa Cells

FEBRUARY 2014 AB70 Abstracts S A T U R D A Y 248 Evaluation of a Novel Missense Activation-Induced Deaminase AID Mutation in a Child with Hyper IgM Syndrome: Is it a Pathogenic Mutation? Dr. Ottavia M. Delmonte, MD, Dr. Feilong Meng, PhD, Dr. Frederick Alt, PhD, Dr. Luigi D. Notarangelo, MD, Dr. Jolan E. Walter, MD, PhD; Pediatric Residency Program, Boston Children’s Hospital, Harvard Medical School, Boston, MA, Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine and Immune Disease Institute, Boston Children’s Hospital, Department of Genetcis, Harvard Medical School, Boston, MA, Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine and Immune Disease Institute, Boston Children’s Hospital, Department of Genetics, Harvard Medical School, Boston, MA, Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, MA; The Manton Center for Orphan Disease Research, Boston Children’s Hospital, Boston, MA, Division of Allergy/Immunology, Massachusetts General Hospital for Children, Harvard Medical School, Boston, MA. RATIONALE: Hyper IgM Syndrome (HIGM) is a primary immunodeficiency associated with defects of CD40, CD40L, AID and UNG genes. In the case of a novel mutation, genetic testing requires secondary confirmation such as functional studies to establish a link between the clinical phenotype and the mutation identified. METHODS: Genetic screening of a 4-year-old boy with clinical features of HIGM Syndrome resulted in a novel missense mutation in the AID gene (p.Ala132Pro). The mutation was located away from the core catalytic domain, but in an area highly conserved among species. The effect of the mutation on AID expression or function was unclear. Testing was undertaken to connect the novel mutation to an actual defect in AID, in vivo and in vitro. In vitro studies included compliment class switch recombination (CSR) assay in AICDA-deficient murine B cells andWestern blot to detect protein stability. AID function in vivo was assessed by IgG level and switched memory B cell fraction. RESULTS: Functional studies confirmed that the mouse AID A132P mutant cannot support CSR in AICDA-deficient murine B cells with retroviral-delivered AID mutant proteins. The AID mutant results in a dysfunctional unstable protein which cannot be detected with Western blot, suggesting that this conserved amino acid could be involved in protein folding and stability. In vivo, the very low amount of AID resulted in low IgG level (<7 mg/dL) and a low fraction of class switched memory B cells (<2%). CONCLUSIONS: Link should be established with functional studies for novel mutations, especially in case of missense mutations.

Watching Aid Scanning Single Stranded and Transcribed DNA with Single Molecule Resolution

The activation-induced deoxycytidine deaminase (AID) is a member the Apobec family of enzymes that catalyzes dC to dU deamination on ssDNA tri-nucleotide motifs. In B cells, it is required to generate antibody diversity by initiating somatic hypermutation (SHM) in the variable region of immunoglobulin genes and class-switch recombination (CSR) in immunoglobulin switch regions. In turn, SHM and CSR are required to generate high-affinity antibodies that bind and neutralize invading antigens. Thus, AID plays an indispensable role in causing mutational diversity to enhance fitness and optimize the immune response.Here, we have used single-molecule fluorescence resonance energy transfer (smFRET) to visualize co-transcriptional scanning of AID. Our data show that AID can follow an active RNA polymerase directionally and processively with speeds upwards of 200 nt/s. However, transcription-stalling leads to bidirectional scanning in the transcription bubble, which in turn, provides AID the necessary time window to carry out deaminations. In bear ssDNA, AID scanning is slow (∼1 s-1), random and bi-directional. The enzyme remains bound to the ssDNA for ∼250 s on average. During this time, it can scan large (>70 nt) ssDNA regions, and it exhibits 'quasi-localization' near favorable deamination motifs. AID also creases the ssDNA during scanning in a sequence dependant manner.

AID and Caspase 8 Shape the Germinal Center Response through Apoptosis

Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

AID-induced remodeling of immunoglobulin genes and B cell fate.

Survival and phenotype of normal and malignant B lymphocytes are critically dependent on constitutive signals by the B cell receptor (BCR) for antigen. In addition, either antigen ligation of the BCR or various mitogenic stimuli result in B cell activation and induction of activation-induced deaminase (AID). AID activity can in turn mediate somatic hypermutation (SHM) of immunoglobulin (Ig) V regions and also deeply remodel the Ig heavy chain locus through class switch recombination (CSR) or locus suicide recombination (LSR). In addition to changes linked to affinity for antigen, modifying the class/isotype (i.e. the structure and function) of the BCR or suddenly deleting BCR expression also modulates the fate of antigen-experienced B cells.

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes.

Alternative End-Joining and Classical Nonhomologous End-Joining Pathways Repair Different Types of Double-Strand Breaks during Class-Switch Recombination

Classical nonhomologous end-joining (C-NHEJ) and alternative end-joining (A-EJ) are the main DNA double-strand break (DSB) repair pathways when a sister chromatid is not available. However, it is not clear how one pathway is chosen over the other to process a given DSB. To address this question, we studied in mouse splenic B cells and CH12F3 cells how C-NHEJ and A-EJ repair DSBs initiated by the activation-induced deaminase during IgH (Igh) class-switch recombination (CSR). We show in this study that lowering the deamination density at the Igh locus increases DSB resolution by microhomology-mediated repair while decreasing C-NHEJ activity. This process occurs without affecting 53BP1 and γH2AX levels during CSR. Mechanistically, lowering deamination density increases exonuclease I recruitment and single-stranded DNA at the Igh locus and promotes C-terminal binding protein interacting protein and MSH2-dependent DSB repair during CSR. Indeed, reducing activation-induced deaminase levels increases CSR efficiency in C-NHEJ-defective cells, suggesting enhanced use of an A-EJ pathway. Our results establish a mechanism by which C-NHEJ and this C-terminal binding protein interacting protein/MSH2-dependent pathway that relies on microhomology can act concurrently but independently to repair different types of DSBs and reveal that the density of DNA lesions influences the choice of DSB repair pathway during CSR.

Large‐scale analysis of somatic hypermutations in antibodies reveals which structural regions, positions and amino acids are modified to improve affinity

The principles of affinity maturation of antibodies (Abs), which underlies Bcell-mediated immunity, are still under debate. It is unclear whether the antigen (Ag) binding site is a preferred target for mutations, and what the role of activation-induced deaminase (AID) hotspots is in this process. Here we report a structural analysis of 3495 residues that have been replaced through somatic hypermutations (SHMs) in 196 Abs. We show that there is no correlation between the propensity of an amino acid to be in AID hotspot and the probability that it is replaced during the SHM process. Although AID hotspots may be necessary to enable SHMs, they are not a major driving force in determining which residues are mutated. We identified Ab positions that are highly mutated and significantly affect binding. The effect of mutation on binding energy is a major factor in determining which structural regions of the Ab are mutated. There is a clear preference for mutations at the Ag-binding site. However, positions outside this region that also affect binding are often preferred targets for SHMs. As for amino acid preferences, a general trend during SHM is to make Ab-Ag interfaces more similar to protein-protein interfaces in general. In different regions of the Ab, there are different sets of preferences for amino acid substitution. This mapping improves our understanding of Ab affinity maturation and may assist in Ab engineering.

Evaluation of IGHV Ultra-Deep Sequences for Activation-Induced Deaminase Characteristics in CLL Cells after T Cell Stimulation

Ultra-deep sequencing has revolutionized our ability to acquire large amounts of genetic data. We have applied this technology towards understanding the mutational process in B-cell chronic lymphocytic leukemia (CLL), which may be a key to understanding CLL pathogenesis. Acquisition of new cytogenetic aberrations and gene mutations in the CLL clone is associated with worse patient outcome. CLL is not unique in this aspect, as new somatic mutations and DNA rearrangements are also found during the evolution of other solid and liquid tumors. In many of these, activation-induced deaminase (AID), an enzyme normally expressed in germinal center B lymphocytes to induce IGHV-D-J mutations and isotype class switch recombination, is abnormally expressed. Its mutational activity, acting outside of the Ig loci, is implicated in the evolution to more aggressive disease.In CLL, the detection of leukemic cells expressing AID ex vivo correlates with significantly shorter patient survival. To test if AID mutational activity is functional in CLL cells and therefore could contribute to CLL evolution, we analyzed mutations in IGHV-D-J, the preferred substrate for AID. Because the rate of AID-induced mutation is low and only a small percentage of CLL cells express AID ex vivo, we used ultra-deep sequencing to analyze CLL cells that were activated under conditions that simulate the CLL microenvironment. Specifically, CLL cells were activated (1) in vitro by simulating the provision of T-cell help or (2) in vivo after adoptive transfer into alymphoid recipient mice, which requires the presence of T-cells for CLL cell growth. Each of these conditions induce AID in a large fraction of CLL cells.To analyze IGHV-D-J mutations, the specific CLL clone IGHV was amplified from cDNA obtained on day 0 or from the activated CLL samples using IGHV family-specific and IGHM primers to enable subsequent comparison of IGHV-D-J with IGHM mutation frequencies. Three unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (>2% compared to germline) CLL (M-CLL) samples were sequenced with the Roche 454 FLX system, resulting in a total of 1,367,522 sequence reads.After using the Roche 454 algorithm to trim sequence reads, they were prepared using custom R scripts that separated 5'IGHV and 5'IGHM primer sequences, aligned sequences to the CLL clone IGHV-D-J rearrangement, and removed poor quality (<20) sequences, insertions, and deletions. Beginning at the 5' end, the script also extracted blocks of sequences of the same length for day 0 and activated samples, which are required for subsequent analyses. After these preparations, the resulting 724,855 sequence blocks were subjected to clonal analyses with custom R scripts. The dominant CLL clone accounted for 94.5% (684,691) of the sequences. Subclone sequences occurring more than once were extracted. After comparison to day 0, new subclones could be identified in all samples after activation (3.22 – 28.70 new subclones / read bp *106).To evaluate AID mutational characteristics in new subclones, SHMTool (http://scb.aecom.yu.edu/shmtool) was employed to calculate mutation frequencies in IGHV-D-J relative to the IGHM constant region, at AID mutation hotspot sites (GYW or WRC), at AID mutation coldspot sites (SYC or GRS), at C/G base pairs, and at error-prone DNA polymerase eta repair hotspot sites (WA or TW). To calculate statistical significance, we utilized a custom R script that used a bootstrap method to account for the large sample sizes provided by ultra-deep sequencing as well as to correct for differences in sequencing sample size.All samples showed an increase in IGHV-D-J versus IGHM mutations after T cell activation. Five of 6 cases showed an increase in AID hostpot mutation frequency. AID coldspot mutation frequency decreased in 3/6 CLL cases. Percent transition mutation at C/G sites was higher than random in 2/6 CLL cases, which correlated with low frequencies of DNA polymerase eta hotspot mutation. In the other 4/6 CLL cases, the lower percent transitions at C/G sites may reflect the contribution of error-prone DNA repair.In summary, we developed a method to analyze ultra-deep IGHV-D-J sequences that revealed AID mutational characteristics in both U-CLL and M-CLL cells after activation with T-cell help in vitro or in vivo. These data are consistent with the hypothesis that AID, perhaps along with error-prone DNA repair, creates new mutations leading to the evolution of aggressive CLL. Disclosures:Rai:Sanofi: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

B-Cell Receptor Selection Pattern and Natural History Of Follicular Lymphoma

Follicular lymphoma (FL) is initiated by the t(14:18) that places the anti-apoptotic proto-oncogene BCL2 under transcriptional control of the immunoglobulin heavy chain (IgH) locus during primary VDJ recombination. Neoplastic FL B cells are arrested at the germinal center stage with ongoing somatic hypermutation of B-cell receptor (BCR) genes by activation-induced deaminase (AID). Antigen recognition by the clonal BCR may facilitate malignant growth upon entry of t(14;18)-immortalized naïve B cells into secondary lymphoid organs. We have reported at ASH 2012 that FL may be classified according to the AID-mediated BCR evolution pattern into two distinct subgroups: A predominantly IgM-expressing subgroup with evidence for antigen-driven affinity maturation, and a subgroup selected for BCR sequence preservation that has often undergone class switch recombination.To investigate whether these BCR selection patterns were correlated to clinical outcome, we retrospectively analyzed somatic hypermutation of multiple BCR sequences in 66 patients (median age: 49; range: 29-75 years) with advanced-stage FL that had undergone a research tumor rebiopsy after inclusion into our prospective idiotype vaccination program (Blood, 2011) with informed consent. Biopsies were performed at a median interval of 20.1 months from diagnosis (range: 0-171 months). At rebiopsy, 43 patients had not yet received systemic cytoreductive therapy (chemotherapy, antibody therapy, or radiation therapy); 14 and 9 patients had been treated with one or two prior regimens, respectively.IgH transcripts were cloned with an unbiased anchored PCR strategy with nested constant region-specific primers. A median of 8 clonal IgH sequences was subjected to bioinformatic analysis for BCR selection patterns by the focused test (Hershberg et al., 2008). 22 patients (33.3%) were classified as having evidence (focused test: p>0) for positive BCR selection through antigen-driven affinity maturation; 44 patients (66.6%) belonged to the BCR preservation category defined by p<0. Clinical risk according to the FLIPI was evenly distributed among both groups (BCR selection: 5 cases low FLIPI, 15 intermediate, 2 high; BCR preservation: 15, 22, 7). The BCR category distribution of app. 1:2 did not change with increasing time from diagnosis to biopsy or after prior therapy.Among 39 patients who were managed initially with a “watch and wait” policy (36 of these underwent rebiopsy prior to initiation of therapy), BCR selection was associated with superior progression-free survival with a median of 87.7 months versus 16.9 months in the BCR preservation group (log-rank test: p=0.024). The treatment-free interval during which the rebiopsy was taken was longer in the BCR selection category for all patients (median 102.0 versus 31.0 months; p=0.030) and for patients with initial “watch and wait” policy (median 103.4 versus 21.0 months; p=0.049). Transformations to aggressive lymphoma occurred exclusively in the BCR preservation group (n=4). At a median total follow-up from diagnosis of 101.6 months, 8 patients (36,4%) in the BCR selection group and 7 patients (15,9%) of the BCR preservation group have not yet received any cytoreductive therapy. When death or transformation were defined as competing events, median event-free survival was not reached in either group with 2 events in the BCR selection group and 8 events in the BCR preservation group (p=0.10).The BCR selection pattern as defined by the focused test through analysis of multiple BCR sequences may represent a novel prognostic factor for the natural history of FL in the pre-treatment phase. A focused test-based categorization could be readily incorporated into the diagnostic work-up of FL and could potentially complement clinical prognostication by means of the FLIPI score. Since the retrospective design of this study cannot entirely exclude possible selection bias, the hypothesis that antigen-driven BCR affinity maturation defines a favorable FL subgroup calls for validation in a prospective study. The relationship of the BCR selection pattern with the established prognostic role of the tumor microenvironment warrants investigation. In summary, we propose that BCR selection may govern the natural history of follicular lymphoma in treatment-free periods, thereby lending further support to the concept that BCR signaling plays an important causal role in FL lymphomagenesis. Disclosures:No relevant conflicts of interest to declare.

Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny

V(D)J joining is mediated by RAG recombinase during early B-lymphocyte development in the bone marrow (BM). Activation-induced deaminase initiates isotype switching in mature B cells of secondary lymphoid structures. Previous studies questioned the strict ontological partitioning of these processes. We show that pro-B cells undergo robust switching to a subset of immunoglobulin H (IgH) isotypes. Chromatin studies reveal that in pro-B cells, the spatial organization of the Igh locus may restrict switching to this subset of isotypes. We demonstrate that in the BM, V(D)J joining and switching are interchangeably inducible, providing an explanation for the hyper-IgE phenotype of Omenn syndrome.

Activation-Induced Deaminase-Coupled DNA Demethylation Is Not Crucial for the Generation of Induced Pluripotent Stem Cells

DNA methylation constitutes a major obstacle in the reprogramming of cells to pluripotency. Although little is known regarding the molecular mechanisms of DNA demethylation, activation-induced deaminase (AID), which is known to function in antibody diversification, has been implicated in DNA demethylation through a base excision repair (BER)-mediated pathway. Here we comprehensively examine the plausibility of coupled AID-BER demethylation in the generation of induced pluripotent stem cells (iPSCs) and show that AID is dispensable for reprogramming cells into iPSCs. Additionally, the overexpression of AID and other factors involved in AID-coupled DNA demethylation does not increase the efficiency of reprogramming. Moreover, BER is not likely to play a role in this process. Our results indicate that the reactivation of key genes governing the pluripotency circuitry occurs through a mechanism that is independent of deamination-coupled demethylation.

AID-Expressing Germinal Center B Cells Cluster Normally within Lymph Node Follicles in the Absence of FDC-M1+ CD35+ Follicular Dendritic Cells but Dissipate Prematurely

Upon activation with T-dependent Ag, B cells enter germinal centers (GC) and upregulate activation-induced deaminase (AID). AID(+) GC B cells then undergo class-switch recombination and somatic hypermutation. Follicular dendritic cells (FDC) are stromal cells that underpin GC and require constitutive signaling through the lymphotoxin (LT) β receptor to be maintained in a fully mature, differentiated state. Although it was shown that FDC can be dispensable for the generation of affinity-matured Ab, in the absence of FDC it is unclear where AID expression occurs. In a mouse model that lacks mature FDC, as well as other LT-sensitive cells, we show that clusters of AID(+)PNA(+)GL7(+) Ag-specific GC B cells form within the B cell follicles of draining lymph nodes, suggesting that FDC are not strictly required for GC formation. However, later in the primary response, FDC-less GC dissipated prematurely, correlating with impaired affinity maturation. We examined whether GC dissipation was due to a lack of FDC or other LTβ receptor-dependent accessory cells and found that, in response to nonreplicating protein Ag, FDC proved to be more critical for long-term GC maintenance. Our study provides a spatial-temporal analysis of Ag-specific B cell activation and AID expression in the context of a peripheral lymph node that lacks FDC-M1(+) CD35(+) FDC and other LT-sensitive cell types, and reveals that FDC are not strictly required for the induction of AID within an organized GC-like environment.

Effects of prostaglandin E2on p53 mRNA transcription and p53 mutagenesis during T‐cell‐independent human B‐cell clonal expansion

Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM(+)IgD(+)CD27(-) B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID(+) cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5 × 10(-4)) greater than the baseline error rate (<0.8 × 10(-4)). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.