Activation-induced Deaminase Research Articles

B-cell tumor development in Tet2-deficient mice

AbstractThe TET2 gene encodes an α-ketoglutarate–dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell–specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2−/− malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA–specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda–deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A–induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

Post-transcriptional regulation of LINE-1 retrotransposition by AID/APOBEC and ADAR deaminases.

Long interspersed element-1 (LINE-1 or L1) retrotransposons represent the only functional family of autonomous transposable elements in humans and formed 17% of our genome. Even though most of the human L1 sequences are inactive, a limited number of copies per individual retain the ability to mobilize by a process termed retrotransposition. The ongoing L1 retrotransposition may result in insertional mutagenesis that could lead to negative consequences suchas genetic disease and cancer. For this reason, cells have evolved several mechanisms of defense to restrict L1 activity. Among them, a critical role for cellular deaminases [activation-induced deaminase (AID)/apolipoprotein B mRNA-editing catalytic polypeptide-like (APOBEC) and adenosine deaminases that act on RNA (ADAR) enzymes] has emerged. The majority of the AID/APOBEC family of proteins are responsible for the deamination of cytosine to uracil (C-to-U editing) within DNA and RNA targets. The ADARs convert adenosine bases to inosines (A-to-I editing) within double-stranded RNA (dsRNA) targets. This review will discuss the current understanding of the regulation of LINE-1 retrotransposition mediated by these enzymes.

A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

Co-Stimulation of BCR and Toll-Like Receptor 7 Increases Somatic Hypermutation, Memory B Cell Formation, and Secondary Antibody Response to Protein Antigen.

The goal of immunization is to produce both a flood of antibodies to neutralize antigen and memory cells to accelerate the secondary response. To enhance the generation of memory B cells, we examined the effect of co-engaging BCR and toll-like receptor (TLR) 7 receptors by immunizing mice with a hapten-protein antigen, NP-CGG, and a ligand, R837 (imiquimod). During the early and late primary responses, there was no augmentation with R837 on the number of germinal center B cells or serum antibody. However, in the niche of germinal centers, R837 increased somatic hypermutation in the canonical VH1-72 gene that encodes NP-specific antibody. Increased mutation was not due to enhanced expression of activation-induced deaminase, but was likely a result of selection for high-affinity B cells with altered codons in the gene. This correlated with the appearance of antigen-specific B cells with a memory phenotype, which was intrinsic to TLR7 on B cells. To determine if these memory cells produced a recall response after a secondary challenge, spleen cells from mice that were immunized with NP-CGG and R837 were adoptively transferred into muMT recipients, and boosted with NP-CGG. Cells from mice that initially received both antigen and R837 generated a robust increase in germinal center B cells, plasmablasts, plasma cells, and serum antibody, compared with their cohorts who received antigen alone. These results support the use of co-immunization with TLR7 ligands to promote vigorous memory B cell responses to protein antigens.

Progesterone attenuates hypertension and autoantibody levels to the angiotensin II type 1 receptor in response to elevated cadmium during pregnancy

IntroductionPreeclampsia is associated with the presence of pathogenic angiotensin-receptor-activating autoantibodies. Cadmium is an increasingly prevalent environmental pollutant that can mimic oestrogens, which may enhance immunoglobulin production. Progesterone exerts opposite effects to oestrogen. MethodsWe measured the levels of cadmium and progesterone in preeclamptic patients and controls. Pregnant rats exposed to cadmium (0.125 mg/kg body weight) from gestational day 9–12 were treated with/without progesterone (3 mg/kg) beginning from gestational day 9 to delivery. We analysed the main features of preeclampsia and circulating level of the angiotensin II type 1 receptor agonistic autoantibody. We also measured the expression of activation-induced cytosine deaminase in B cells. ResultsThere were higher cadmium levels and lower progesterone levels in the blood of preeclamptic women than in the blood of those with a healthy pregnancy. Based on this finding, a rat model of preeclampsia was established by intraperitoneally administrating low-dose cadmium on gestational days 9–12. Rats were then treated with/without progesterone. Key features of preeclampsia, including hypertension, proteinuria and placental abnormalities, appeared in pregnant rats after cadmium injection and improved after treatment with progesterone. Cadmium increased immunoglobulin production, mainly angiotensin II type 1-receptor-agonistic autoantibodies, by increasing the expression of activation-induced cytosine deaminase in B cells; progesterone exerted an opposite effect. ConclusionCadmium induced immune abnormalities that may be a key pathogenic contributor to preeclampsia. Progesterone supplementation to correct hormonal imbalance may be a viable strategy for preeclampsia management.

The role of Nuclear Factor-kappa B signaling in human cervical cancer.

Background The Nuclear Factor kappaB (NF-kB) family consists of transcription factors that play a complex and essential role in the regulation of immune responses and inflammation. NF-kB has recently generated considerable interest as it has been implicated in human cancer initiation, progression and resistance to treatment. In the present comprehensive review the different aspects of NF-kB signaling in the carcinogenesis of cancer of the uterine cervix are discussed. NF-kB functions as part of a network, which determines the pattern of its effects on the expression of several other genes (such as crosstalks with reactive oxygen species, p53, STAT3 and miRNAS) and thus its function. Activation of NF-kB triggered by a HPV infection is playing an important role in the innate and adaptive immune response of the host. The virus induces down regulation of NF-kB to liquidate the inhibitory activity for its replication triggered by the immune system leading a status of persistant HPV infection. During the progression to high grade intraepithelial neoplasia and cervical cancer NF-KB becomes constitutionally activated again. Mutations in NF-kB genes are rare in solid tumors but mutations of upstream signaling molecules such as RAS, EGFR, PGF, HER2 have been implicated in elevated NF-kB signaling. NF-kB can stimulate transcription of proliferation regulating genes (eg. cyclin D1 and c-myc), genes involved in metastasis, VEGF dependent angiogenesis and cell immortality by telomerase. NF-kB activation can also induce the expression of activation-induced cytodine deaminase (AID) and the APOBEC proteins, providing a mechanistic link between the NF-kB pathway and mutagenic characteristic of cervical cancer. Inhibition of NF-kB has the potential to be used to reverse resistance to radiotherapy and systemic anti-cancer medication, but currently no clinicaly active NF-kB targeting strategies are available.

A novel class of chemicals that react with abasic sites in DNA and specifically kill B cell cancers.

Most B cell cancers overexpress the enzyme activation-induced deaminase at high levels and this enzyme converts cytosines in DNA to uracil. The constitutive expression of this enzyme in these cells greatly increases the uracil content of their genomes. We show here that these genomes also contain high levels of abasic sites presumably created during the repair of uracils through base-excision repair. We further show that three alkoxyamines with an alkyne functional group covalently link to abasic sites in DNA and kill immortalized cell lines created from B cell lymphomas, but not other cancers. They also do not kill normal B cells. Treatment of cancer cells with one of these chemicals causes strand breaks, and the sensitivity of the cells to this chemical depends on the ability of the cells to go through the S phase. However, other alkoxyamines that also link to abasic sites- but lack the alkyne functionality- do not kill cells from B cell lymphomas. This shows that the ability of alkoxyamines to covalently link to abasic sites is insufficient for their cytotoxicity and that the alkyne functionality may play a role in it. These chemicals violate the commonly accepted bioorthogonality of alkynes and are attractive prototypes for anti-B cell cancer agents.

Accelerated Systemic Autoimmunity in the Absence of Somatic Hypermutation in 564Igi: A Mouse Model of Systemic Lupus with Knocked-In Heavy and Light Chain Genes.

564Igi mice have knocked-in immunoglobulin (Ig) heavy (H) and light (L) chain genes that encode an autoantibody recognizing RNA. Previously, we showed that these mice produce pathogenic IgG autoantibodies when activation-induced deaminase (AID) is expressed in pre-B and immature B cells but not when it is expressed only in mature B cells. AID has two functions; it is necessary for somatic hypermutation (SHM) and class switch recombination (CSR). To determine the role of each of these functions in the generation of pathogenic autoantibodies, we generated 564Igi mice that carry a mutant AID-encoding gene, Aicda (AicdaG23S), which is capable of promoting CSR but not SHM. We found that 564Igi AicdaG23S mice secreted class-switched antibodies (Abs) at levels approximately equal to 564Igi mice. However, compared to 564Igi mice, 564Igi AicdaG23S mice had increased pathogenic IgG Abs and severe systemic lupus erythematosus-like disease, including, glomerulonephritis, and early death. We suggest that in 564Igi mice SHM by AID changes Ig receptors away from self reactivity, thereby mitigating the production of autoantibody, providing a novel mechanism of tolerance.

Abstract 3802: A novel uracil-DNA glycosylase, UdgX, as a new biochemical tool to directly detect uracils in DNA

Abstract The enzyme activation-induced deaminase (AID) is essential to the B lymphocyte development and antibody maturation. It converts cytosines in DNA to uracil. The unregulated expression of AID in many B cell cancers is correlated with an increased load of mutations. AID also promotes DNA strand breaks and is required for chromosomal translocations such as IgH/Myc in these tumors. We have previously showed that B cell cancer cell lines and patient tumors which express AID at high levels also have high levels of uracil in their genomic DNA. We accomplished this by excising the uracils using a uracil-DNA glycosylase (Ung) and labeled the resulting in an abasic site with an aldehyde reactive chemical (ARP). ARP contains a biotin tag and it was detected using a fluorescently labeled streptavidin. The results from this assay showed B cell tumors have as much as 100-fold higher levels of genomic uracils. Further development of this assay has allowed the detection of uracils in the blood plasma and buffy coat of B cell cancer patients. This “liquid biopsy” assay technique has the potential to work as a simple surveillance tool for B cell cancer patients who are in remission. In addition, we are exploring the possibility of a direct detection of uracil in DNA, which would provide greater sensitivity to this assay and simplify it. A unique uracil-DNA glycosylase was recently isolated from M. smegmatis, UdgX, that binds covalently to uracils in DNA. We are developing multiple ways to detect uracil-DNA-UdgX complexes and amplifying the resulting signal. We expect that this will allow us to directly visualize uracils in B-NHL tumors and detect uracils in circulating B-NHL tumor DNA from a drop of patient plasma. Citation Format: Jessica Stewart, Shanqiao Wei, Madhurima Datta, Umesh Varshney, Ashok Bhagwat. A novel uracil-DNA glycosylase, UdgX, as a new biochemical tool to directly detect uracils in DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3802. doi:10.1158/1538-7445.AM2017-3802

R Loops in the Regulation of Antibody Gene Diversification.

For nearly three decades, R loops have been closely linked with class switch recombination (CSR), the process that generates antibody isotypes and that occurs via a complex cascade initiated by transcription-coupled mutagenesis in switch recombination sequences. R loops form during transcription of switch recombination sequences in vitro and in vivo, and there is solid evidence that R loops are required for efficient class switching. The classical model of R loops posits that they boost mutation rates by generating stable and long tracts of single-stranded DNA that serve as the substrate for activation induced deaminase (AID), the enzyme that initiates the CSR reaction cascade by co-transcriptionally mutating ssDNA in switch recombination sequences. Though logical and compelling, this model has not been supported by in vivo evidence. Indeed, several reports suggest that R loops may not be involved in recruiting AID activity to switch regions, meaning that R loops probably serve other unanticipated roles in CSR. Here, I review the key findings in this field to date and propose hypotheses that could help towards elucidating the precise function of R loops in CSR.

APOBEC gene family expression and hallmarks in chronic lymphocytic leukemia

Abstract The hallmark activity of APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) family of cytidine deaminases, including activation-induced deaminase (AID) and APOBEC3 genes, has been detected in somatic mutation signatures by ultra-deep sequencing of the genomes of many cancers, including chronic lymphocytic leukemia (CLL). The acquisition of these mutations is hypothesized to lead to the progression towards aggressive disease in cancer. To examine this in CLL, we tested if increased APOBEC family member gene expression in CLL cells, as measured by microarray and quantitative real time PCR, correlated with worse patient outcome. Higher levels of AID, APOBEC3B, APOBEC3F and APOBEC3H in CLL cells correlated with worse patient outcome, whereas APOBEC3G did not. Interestingly, higher levels of a truncated splice variant of APOBEC3F tended to correlate with better patient outcome. The expression of truncated APOBEC3F may possibly interfere with APOBEC family member mutation activity. To test mutation activity, CLL cells were activated by transfer into NOD-scid IL2Rγnull mice, a xenograft model of human CLL, and hallmark mutation signatures in the expressed immunoglobulin variable region (IGHV) of CLL cells were analyzed by targeted ultra-deep sequencing. Induced IGHV mutation hallmarks consistent with AID were found. These data support the hypothesis that expression and mutation activity of APOBEC family members, such as AID, in CLL cells could lead to adverse patient consequences.

Inhibiting a mutagen: mitigating Activation-Induced Deaminase activity

Abstract Activation-Induced Deaminase (AID) is a cytosine deaminase which converts cytosine into uracil within stretches of ssDNA. The presence of uracil in DNA initiates a cascade of mutagenic DNA repair to introduce point mutations and DNA double strand breaks. Specific targeting of AID to the immunoglobulin heavy chain loci (Igh) promotes mutagenesis in the antibody variable region for somatic hypermutation (SHM) or switch regions leading to class switch recombination (CSR). However, AID also plays a role in accumulating unnecessary mutations across the genome. Mis-targeting of AID results in the initiation of tumor development and leads to greater drug resistance among cancer cells when continually expressed. To identify an AID inhibitor, we screened ~400,000 compounds in conjunction with the NCATS core facility at NIH. Using FRET based analysis of cytosine deamination, we have identified approximately 150 potential inhibitors of AID catalytic activity. To confirm biological function, we have examined the compounds’ effects on CSR in an in vitro murine B-cell activation assay. Of 30 confirmed inhibitor candidates, we have selected the top 7 to proceed with further characterization to determine if they prevent uracil accumulation in primary mouse cells after ex vivo stimulation. Using these approaches, we hope to identify a small molecule with great efficacy and low toxicity for use as a molecular probe to further characterize AID’s intricacies, or even as a therapeutic.

The Number of Overlapping AID Hotspots in Germline IGHV Genes Is Inversely Correlated with Mutation Frequency in Chronic Lymphocytic Leukemia.

The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01. An absence of V-region mutations in CLL patients (“IGHV unmutated”, or U-CLL) is associated with a poorer prognosis compared to “IGHV mutated” (M-CLL) patients. The reasons for this difference are still unclear, but it has been noted that particular IGHV genes associate with U-CLL vs M-CLL. For example, patients with IGHV1-69 clones tend to be U-CLL with a poor prognosis, whereas patients with IGHV3-30 tend to be M-CLL and have a better prognosis. Another distinctive feature of CLL is that ~30% of (mostly poor prognosis) patients can be classified into “stereotyped” subsets, each defined by HCDR3 similarity, suggesting selection, possibly for a self-antigen. We analyzed >1000 IGHV genes from CLL patients and found a highly significant statistical relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in patients. However, paradoxically, this correlation was inverse, with V-regions with more WGCW hotspots being less likely to be mutated, i.e., more likely to be U-CLL. The number of WGCW hotspots in particular, are more strongly correlated with mutation frequency than either non-overlapping (WRC) hotspots or more general models of mutability derived from somatic hypermutation data. Furthermore, this correlation is not observed in sequences from the B cell repertoires of normal individuals and those with autoimmune diseases.

R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching

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.

Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination.

In order to promote an efficient humoral immune response, germinal center B cells modify both the antigen recognition and effector domains by programmed genetic alterations of their antibody genes. To do so, B cells use the enzyme activation-induced deaminase (AID), which transforms deoxycytidine into deoxyuridine at the immunoglobulin genes, triggering mutagenic DNA repair. Data accumulated during the past decade have significantly advanced our understanding of how AID activity is regulated and preferentially targeted to the immunoglobulin genes. There is also a better understanding of the ways by which AID-catalyzed uracil is recognized and the ensuing downstream processing underpinning the mechanisms of somatic hypermutation and class switch recombination. Here, we critically review these advances in the context of their relevance for the humoral immune response. A detailed understanding of these molecular mechanisms is paramount to uncover the basis of B cell intrinsic immunodeficiency, as well as to suggest tools and strategies that might allow boosting antibody gene diversification in the context of immunizations or infections that require the elicitation of rare or highly mutated antibody variants.

Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE.

Noncoding transcripts originating upstream of the immunoglobulin constant region (I transcripts) are required to direct activation-induced deaminase to initiate class switching in B cells. Differential regulation of Iε and Iγ1 transcription in response to interleukin 4 (IL-4), hence class switching to IgE and IgG1, is not fully understood. In this study, we combine novel mouse reporters and single-cell RNA sequencing to reveal the heterogeneity in IL-4-induced I transcription. We identify an early population of cells expressing Iε but not Iγ1 and demonstrate that early Iε transcription leads to switching to IgE and occurs at lower activation levels than Iγ1. Our results reveal how probabilistic transcription with a lower activation threshold for Iε directs the early choice of IgE versus IgG1, a key physiological response against parasitic infestations and a mediator of allergy and asthma.

Activation induced deaminase mutational signature overlaps with CpG methylation sites in follicular lymphoma and other cancers.

Follicular lymphoma (FL) is an uncurable cancer characterized by progressive severity of relapses. We analyzed sequence context specificity of mutations in the B cells from a large cohort of FL patients. We revealed substantial excess of mutations within a novel hybrid nucleotide motif: the signature of somatic hypermutation (SHM) enzyme, Activation Induced Deaminase (AID), which overlaps the CpG methylation site. This finding implies that in FL the SHM machinery acts at genomic sites containing methylated cytosine. We identified the prevalence of this hybrid mutational signature in many other types of human cancer, suggesting that AID-mediated, CpG-methylation dependent mutagenesis is a common feature of tumorigenesis.

Alternative Splicing of APOBEC3D Generates Functional Diversity and Its Role As a DNA Mutator

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family consists of 11 members: APOBEC1, APOBEC2, seven APOBEC3s (A3s) (A/B/C/D/F/G/H), APOBEC4, and the activation-induced deaminase (AID). APOBEC1, A3s, and AID have cytidine deaminase activity and induce a cytidine (C) to thymidine (T) transition. The main function of A3s is to trigger an innate immune response to viral infection such as human immunodeficiency virus-1. Recently, it was reported that several APOBEC family proteins can induce somatic mutations into genomic DNA and thus promote cancer development. For example, AID-mediated somatic mutations contribute to B-cell lymphoma and A3B can be an enzymatic source in solid tumors such as breast cancer. However, little is known about other A3 member proteins.To determine the expression of A3 in hematopoietic cells, we performed quantitative PCR in leukemia cell lines. A3B, A3C, A3D, A3F and A3G were detected in all cell lines, but not A3A or A3H. Next, we analyzed published The Cancer Genome Atlas data from patients with acute myeloid leukemia to find somatic alterations in A3 genes using cBioPortal (http://www.cbioportal.org/). We found that each A3 was upregulated at 4-7 % of all samples and the total frequency of 23 %. Interestingly, overall survival of patients with A3upregulation was lower than those without upregulation, suggesting an important role of A3s in pathogenesis of leukemia. As it has been shown that A3A, A3B, and A3D show capacity to inflict DNA damage, we decided to investigate whether A3D can induce mutations in foreign and genomic DNA.During our attempt to generate A3D expression constructs, we detected multiple bands in leukemia and lung cancer cell lines. Based on our analysis and previous reports, we confirmed that there are at least 7 transcript variants (v1 to 7). Because A3D v3, v4 and v5 lack cytidine deaminase domains, we decided to examine A3Dv1, v2, v6 and v7.To determine whether A3D variants have foreign DNA editing activity, we performed differential DNA denaturation (3D)-PCR in HEK293T cells co-transfected with expression vectors for EGFP, UNG inhibitor, and pCAG-GS containing A3Dv1, v2, v6, v7, A3B or empty control. Total DNA was isolated 4 days after transfection. PCR products were detected at lower denaturation temperature (Td) in cells expressing A3B and all A3D variants vectors compared to control. Sequences of PCR products at lowest Td revealed that mutation frequencies in EGFP gene were 6.1-10.5 per 103 bps in cells transfected with all A3D variants and 18.7 per 103bps in cells transfected with A3B. Of note, 90 % of mutations were C/G to T/A transition in cells transfected with A3B, but in cells transfected with A3D these were 30-60 %.Next we performed 3D-PCR in HEK293T cells retrovirally infected with MigR1-IRES-EGFP containing A3Dv1, v2, v6, v7, A3B or empty to determine genomic DNA editing activity. Total DNA was isolated 4 weeks after infection. At lower Td, PCR products were detected only in cells expressing A3Dv1 and A3B. PCR products in cells expressing A3Dv2, v6, v7 were detected only at same Td as control. Sequences of the PCR products at lowest Td revealed that the mutation frequency in the EGFP gene was 22.2 per 103 bps in cells expressing A3Dv1, and 56.9 per 103 bps in cells expressing A3B. However mutation frequencies in cells expressing A3Dv6 or v7 were lower than 2 per 103bps and no mutations were detected in cells expressing A3Dv2. All of these mutations were C/G to T/A transition. Interestingly, we found 4 clones with deletion in cells expressing A3Dv1, v6 and A3B. In addition, all clones had several bases of micro homologous sequences in broken ends before joining. These results suggested that double strand breaks in genomic DNA were repaired by microhomology-mediated end joining.These DNA editing assays showed that all A3D variants had ability to induce mutations in foreign DNA, and A3Dv1 was major isoform which had ability to induce mutations in genomic DNA.Taken together, our experiments showed that A3D can be a DNA mutator and alternative splicing generates functional diversity of A3D. These findings suggest that A3D may contribute to development of hematopoietic malignancies. DisclosuresTakaori-Kondo:Mochida Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding; Takeda Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Kyowa Kirin: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.

DNA demethylation pathways: Additional players and regulators.

DNA demethylation can occur passively by "dilution" of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-methylcytosine (5mC) to cytosine (C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten-eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well-known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA demethylation. Here, we review these new developments.

B Cells Gasping for Air

Humoral immunity and vaccine efficacy depend on the proliferation, affinity maturation and class switch recombination (CSR) of B cells in germinal centers (GCs). It is well appreciated that the cytokines present in this microenvironment determine the isotype to which differentiating B cells will switch. In this recent manuscript, Cho and colleagues find an important role for another factor, oxygen levels, in modulating the effector function of GC B cells. The proportion of oxygen (pO2) can vary from 21% in the atmosphere, to 5% in the venous blood, and 1% in the depth of certain tissues, such that cells may be exposed to various pO2 levels, depending on where they are in the body. The authors injected into mice chemical probes that mark hypoxic cells and showed that GC B cells were more hypoxic than other B cells in the spleen, and that the most hypoxic cells were found in the light zone of the GC, an area of less intense proliferation than the dark zone (Figure 1). Hypoxic B cells proliferated less and displayed increased apoptotic signaling both in vivo and in a hypoxic chamber in vitro. Hypoxia resulted in decreased switching to IgG1 and IgG2c isotypes (the latter an IFN-γ-driven isotype in C57BL/6 mice, analogous to IgG2a in other strains, and important for antimicrobial and inflammatory responses), even within B cells that had successfully proliferated several cycles. This was true in both primary and secondary immune responses. In contrast, hypoxia minimally affected IgA class switch, perhaps an adaptation to the fact that the intestine, where most IgA is produced, has low pO2 constitutively. Hypoxia also switched the metabolic program of activated B cells by promoting a higher glycolytic rate. Mechanistically, hypoxia reduced expression of activation-induced deaminase (AID, an enzyme that drives CSR) in IgG but not IgA switch conditions. Immunization of mice with conditional deletion of von Hippel–Lindau protein selectively in B cells, a trick to stabilize hypoxia-induced factor (HIF) and mimic hypoxia, recapitulated the HIF-dependent reduction of high affinity IgG1 antibodies and of IgG2c antibodies of all affinities. These defects, which were corrected if expression of AID was forced in B cells, correlated with reduced mammalian target of rapamycin complex 1 (mTORC1) activation. Diminished antibody production was mimicked by Rptr haploinsufficiency (a molecule necessary for mTORC1 activation). Of particular interest to transplant professionals, treatment of B cells with rapamycin also reduced AID levels and IgG2c switching. Therefore, the pO2 to which GC B cells are exposed determines their proliferation, survival, affinity maturation and CSR, and modulating local oxygen levels or influencing the signaling pathways elicited by hypoxia could potentially be a therapeutic strategy to manipulate humoral responses in disease. Alloantibody production in transplantation may be the main cause of chronic rejection and late graft loss, vexing attempts at increasing organs’ half-lives. Whereas primary humoral alloresponses are likely initiated in secondary lymphoid organs, tertiary lymphoid structures may develop over time within the transplanted organ itself. B cells from these structures may respond to cryptic self-antigens and undergo affinity maturation locally. The data from Cho and colleagues suggest that levels of pO2 in transplanted organs should be investigated. Thus, targeting rapamycin to GCs and to the graft, or manipulating oxygen levels locally, may be an approach to reduce initial production of alloantibodies and secondary epitope spreading to cryptic self-antigens. Dr. Alegre is a professor in the Department of Medicine at the University of Chicago.