Activation-induced Cytidine Deaminase Expression Research Articles

Cigarette smoke inhibits BAFF expression and mucosal immunoglobulin A responses in the lung during influenza virus infection.

BackgroundIt is incompletely understood how cigarette smoke (CS) exposure affects lung mucosal immune responses during viral respiratory infections. B cell activating factor belonging to the tumor necrosis factor family (BAFF) plays an important role in the induction of secretory immunoglobulin A (S-IgA) which is the main effector of the mucosal immune system. We therefore investigated the effects of CS exposure on BAFF expression and S-IgA responses in the lung during influenza virus infection.MethodsMice were exposed to CS and/or infected with influenza virus. Bronchoalveolar lavage fluid and lung compartments were analyzed for BAFF expression, influenza-specific S-IgA level and histological changes. Lung B cells were isolated and the activation-induced cytidine deaminase (Aicda) expression was determined. BEAS-2B cells were treated with CS extract (CSE), influenza virus, interferon beta or N-acetylcysteine and BAFF expression was measured.ResultsCS inhibited BAFF expression in the lung, particularly after long-term exposure. BAFF and S-IgA levels were increased during influenza virus infection. Three-month CS exposure prior to influenza virus infection resulted in reduced BAFF and S-IgA levels in the lung as well as augmented pulmonary inflammation on day 7 after infection. Prior CS exposure also caused decreased Aicda expression in lung B cells during infection. Neutralization of BAFF in the lung resulted in reduced S-IgA levels during influenza virus infection. CSE inhibited virus-mediated BAFF induction in a dose-dependent manner in BEAS-2B cells, while this inhibition of BAFF by CSE was prevented by pretreatment with the antioxidant N-acetylcysteine.ConclusionsOur findings indicate that CS may hinder early mucosal IgA responses in the lung during influenza virus infection through oxidative inhibition of BAFF, which might contribute to the increased incidence and severity of viral infections in smokers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0201-y) contains supplementary material, which is available to authorized users.

Chronic lung injury by constitutive expression of activation-induced cytidine deaminase leads to focal mucous cell metaplasia and cancer.

Activation-induced cytidine deaminase (AID) is an enzyme required for antibody diversification, and it causes DNA mutations and strand breaks. Constitutive AID expression in mice invariably caused lung lesions morphologically similar to human atypical adenomatous hyperplasia (AAH), which can be a precursor of bronchioloalveolar carcinoma. Similar to AAH, mouse AAH-like lesion (MALL) exhibited signs of alveolar differentiation, judging from the expression of alveolar type II (AT2) cell marker surfactant protein C (SP-C). However, electron microscopy indicated that MALL, which possessed certain features of a mucous cell, is distinct from an AAH or AT2 cell. Although MALL developed in all individuals within 30 weeks after birth, lung tumors occurred in only 10%; this suggests that the vast majority of MALLs fail to grow into visible tumors. MALL expressed several recently described markers of lung alveolar regeneration such as p63, keratin 5, keratin 14, leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and Lgr6. Increased cell death was observed in the lungs of AID transgenic mice compared with wild-type mice. Based on these observations, we speculate that MALL is a regenerating tissue compensating for cellular loss caused by AID cytotoxicity. AID expression in such regenerating tissue should predispose cells to malignant transformation via its mutagenic activity.

BCL2 mutations are associated with increased risk of transformation and shortened survival in follicular lymphoma

AbstractFollicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma.

Overexpression of Activation-Induced Cytidine Deaminase in MTX- and Age-Related Epstein-Barr Virus-Associated B-Cell Lymphoproliferative Disorders of the Head and Neck.

Recent research has shown that activation-induced cytidine deaminase (AID) triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Such aberrant AID expression is seen in B-cell leukemia/lymphomas, including Burkitt lymphoma which is associated with c-myc translocation. Moreover, Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1) increases genomic instability through early growth transcription response-1 (Egr-1) mediated upregulation of AID in B-cell lymphoma. However, few clinicopathological studies have focused on AID expression in lymphoproliferative disorders (LPDs). Therefore, we conducted an immunohistochemical study to investigate the relationship between AID and LMP-1 expression in LPDs (MTX-/Age-related EBV-associated), including diffuse large B-cell lymphomas (DLBCLs). More intense AID expression was detected in LPDs (89.5%) than in DLBCLs (20.0%), and the expression of LMP-1 and EBER was more intense in LPDs (68.4% and 94.7%) than in DLBCLs (10.0% and 20.0%). Furthermore, stronger Egr-1 expression was found in MTX/Age-EBV-LPDs (83.3%) than in DLBCLs (30.0%). AID expression was significantly constitutively overexpressed in LPDs as compared with DLBCLs. These results suggest that increased AID expression in LPDs may be one of the processes involved in lymphomagenesis, thereby further increasing the survival of genetically destabilized B-cells. AID expression may be a useful indicator for differentiation between LPDs and DLBCLs.

MicroRNA 21 Expression Levels in HIV Negative and HIV Positive Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease with various morphological and molecular subtypes. It commonly occurs in the elderly as well as in people infected with HIV. According to the Hans' algorithm DLBCL can be classified prognostically into the favourable germinal centre (GC) subtype and the unfavourable non- GC subtype. The disease tends to be more aggressive in HIV infected individuals. Research on DLBCL has been largely conducted on HIV negative samples and it is still unclear how HIV affects the molecular mechanisms of the disease. We conducted a study to compare the expression levels of miR-21 in HIV negative and HIV positive DLBCL patients. Our results suggest that miR-21 expression levels are higher in HIV positive cases of DLBCL. We did not observe any statistically significant difference in miR-21 levels between DLBCL subtypes in both HIV negative and positive cases. Favourable prognosis in HIV negative patients was associated with high miR-21 expression and the reverse was true in HIV positive patients. In conclusion, our results indicate that HIV infection may affect the expression of miR-21 in DLBCL. This highlights the need for further studies on HIV positive DLBCL as the prognostic significance of miR-21 expression level may differ depending on the HIV status. Keywords: B-cell lymphoma; Micro RNA; Prognosis; HIV; miR-21; DLBCL Introduction Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) comprising of 30% of all NHL [1]. The disease shows marked heterogeneity, with morphological and molecular variants [2,3], making it difficult to treat. DLBCL is divided into two histological subtypes with prognostic significance according to the Hans' algorithm. The non-germinal centre (non-GC) subtype has a worse prognosis than the germinal centre B cell like (GC) subtype [4]. Recent data suggests that HIV gp120 induces B cells expressing mannose C type lectin receptors (MCLRs) to undergo immunoglobulin class switching by increasing the expression of activation-induced cytidine deaminase [5]. Chronic B-cell activation in HIV infection is driven by the production of B-cell stimulating cytokines which are secreted by gp120 bound monocytes [6]. These cytokines act by upregulating MCLRs on the surface of B cells, thus creating chronic B cell activation [5]. Therefore, people infected with HIV are at an increased risk of developing B cell NHL [7]. DLBCL is the most common NHL diagnosed in HIV infected individuals [8,9]. MicroRNAs are non-coding RNA molecules that silence the expression of target genes [10]. Gene silencing is achieved by imperfect or perfect pairing of microRNA with3’UTR of target mRNA thereby resulting in inhibition of translation or degradation, respectively [11,12]. They are transcribed as long transcripts

The involvement of Streptococcus anginosus infection and the aberrant activation-induced cytidine deaminase (AID) expression in human oral squamous cell carcinoma

The involvement of Streptococcus anginosus infection and the aberrant activation-induced cytidine deaminase (AID) expression in human oral squamous cell carcinoma

Epigenetic function of activation-induced cytidine deaminase and its link to lymphomagenesis.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes during B cell maturation and immune response. Expression of AID is tightly regulated due to its mutagenic and recombinogenic potential, which is known to target not only Ig genes, but also non-Ig genes, contributing to lymphomagenesis. In recent years, a new epigenetic function of AID and its link to DNA demethylation came to light in several developmental systems. In this review, we summarize existing evidence linking deamination of unmodified and modified cytidine by AID to base-excision repair and mismatch repair machinery resulting in passive or active removal of DNA methylation mark, with the focus on B cell biology. We also discuss potential contribution of AID-dependent DNA hypomethylation to lymphomagenesis.

Histone deacetylase inhibitors upregulate B cell microRNAs that silence AID and Blimp-1 expression for epigenetic modulation of antibody and autoantibody responses.

Class-switch DNA recombination (CSR) and somatic hypermutation (SHM), which require activation-induced cytidine deaminase (AID), and plasma cell differentiation, which requires B lymphocyte-induced maturation protein-1 (Blimp-1), are critical for the generation of class-switched and hypermutated (mature) Ab and autoantibody responses. We show that histone deacetylase inhibitors valproic acid and butyrate dampened AICDA/Aicda (AID) and PRDM1/Prdm1 (Blimp-1) mRNAs by upregulating miR-155, miR-181b, and miR-361 to silence AICDA/Aicda, and miR-23b, miR-30a, and miR-125b to silence PRDM1/Prdm1, in human and mouse B cells. This led to downregulation of AID, Blimp-1, and X-box binding protein 1, thereby inhibiting CSR, SHM, and plasma cell differentiation without altering B cell viability or proliferation. The selectivity of histone deacetylase inhibitor-mediated silencing of AICDA/Aicda and PRDM1/Prdm1 was emphasized by unchanged expression of HoxC4 and Irf4 (important inducers/modulators of AICDA/Aicda), Rev1 and Ung (central elements for CSR/SHM), and Bcl6, Bach2, or Pax5 (repressors of PRDM1/Prdm1 expression), as well as unchanged expression of miR-19a/b, miR-20a, and miR-25, which are not known to regulate AICDA/Aicda or PRDM1/Prdm1. Through these B cell-intrinsic epigenetic mechanisms, valproic acid blunted class-switched and hypermutated T-dependent and T-independent Ab responses in C57BL/6 mice. In addition, it decreased class-switched and hypermutated autoantibodies, ameliorated disease, and extended survival in lupus MRL/Fas(lpr/lpr) mice. Our findings outline epigenetic mechanisms that modulate expression of an enzyme (AID) and transcription factors (Blimp-1 and X-box binding protein 1) that are critical to the B cell differentiation processes that underpin Ab and autoantibody responses. They also provide therapeutic proof-of-principle in autoantibody-mediated autoimmunity.

IGHV-D-J Ultra-Deep Sequencing Reveals APOBEC and AID Targeted Mutations during Clonal Evolution of CLL in a Xenograft Mouse Model

Targeted ultra-deep sequencing of chronic lymphocytic leukemia (CLL) cells has enabled the assessment of subclone development based on mutations in the IGHV-D-J signature sequence in the dominant CLL clone. We have utilized the Roche 454 FLX pyrosequencing system, which can generate long sequencing reads containing both the immunoglobulin variable region (IGHV-D-J) and part of the immunoglobulin μ constant region (IGHM) in a single sequence, to analyze the mutational characteristics of newly evolved subclones to determine if they derive from AID/APOBEC activity. APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) is a family of cytidine deaminases that includes AID (activation-induced cytidine deaminase). AID is required for somatic hypermutation in germinal center B lymphocytes. CLL cells, like most non-germinal center B lymphocytes, generally do not express AID. However, AID expression in a small fraction of the CLL clone correlates with worse patient outcome. This observation has led to the hypothesis that abnormal AID expression promotes new off-target non-immunoglobulin mutations and DNA deletions and rearrangements leading to the development of more aggressive disease. CLL is not alone in this hypothesis, as AID is involved in the evolution of other leukemias/lymphomas and reportedly in other types of tumors such as breast and gastrointestinal cancers. Large scale cataloguing of somatic mutations by ultra-deep sequencing of a wide array of cancers has revealed an AID/APOBEC mutational signature in many cancers, including CLL (Alexandrov et al. 2013 Nature). Thus, AID/APOBEC family members may be involved in somatic mutations leading to the evolution of aggressive cancers.To test if AID/APOBEC proteins could be mutationally active in CLL, we analyzed the characteristics of new mutations found in IGHV-D-J-M in CLL cells that were activated in vivo after adoptive transfer into alymphoid NOD/Shi-scid,γcnull (NSG) mice. This CLL xenograft model activates a large fraction of CLL cells, which become AID+ and facilitates the detection of new subclones expressing mutated IGHV-D-Js by ultra-deep sequencing. Four unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (>2% compared to germline) CLL (M-CLL) samples were each adoptively transferred into individual NSG mice. After expansion of CLL, the mice were sacrificed and the specific CLL clone IGHV-D-J-M was amplified from xenograft mouse spleen cDNA. Pre-transfer CLL cell cDNA was also amplified to establish a baseline comparison. Ultra-deep sequencing of these samples resulted in 2,318,800 sequence reads, which were subsequently trimmed according to the Roche 454 algorithm and further processed by custom R scripts. The sequence reads were aligned to the dominant CLL clone IGHV-D-J-M sequence to remove insertions, deletions, and poor quality (<20) nucleotides, resulting in 1,700,839 blocks of sequences of the same length. The dominant CLL clones represented 92.3% (1,569,059 blocks) of these sequences and were excluded. Mutated subclone sequences that occurred at least twice were extracted. Xenograft subclones not found in the pre-transfer sample were selected for analyses of the characteristics of newly generated CLL mutations. New xenograft CLL subclones could be identified in all samples (3.2 – 12.3 new subclones / read bp *106).AID mutational characteristics in new subclones were assessed using the SHMTool (http://scb.aecom.yu.edu/shmtool) algorithms to calculate mutation frequencies in IGHV-D-J relative to IGHM and at AID mutation hotspots and coldspots. Other APOBEC family members have a different mutation hotspot site, which we analyzed by custom R scripts.All CLL samples showed evidence of AID mutational activity: higher IGHV-D-J versus IGHM mutations in all cases, increased AID hotspot mutation frequency in five cases, and decreased AID coldspot mutation frequency in five cases. Increased APOBEC hotspot mutational activity was seen in four cases. This APOBEC mutational activity is consistent with increased APOBEC3 gene expression and the genomic somatic mutation pattern observed recently in CLL (Rebhandl et al. 2014 Leukemia). Thus, both U-CLL and M-CLL are capable of producing mutations characteristic of AID or APOBEC activity. These data are consistent with the hypothesis that AID/APOBEC may promote new mutations leading to the evolution of aggressive CLL. DisclosuresNo relevant conflicts of interest to declare.

HIV-1 induces B-cell activation and class switch recombination via spleen tyrosine kinase and c-Jun N-terminal kinase pathways.

Patients infected by the HIV type 1 (HIV-1) frequently show a general deregulation of immune system. A direct influence of HIV-1 particles on B-cell activation, proliferation and B-cell phenotype alterations has been recently described. Moreover, expression of activation-induced cytidinedeaminase (AID) mRNA, which is responsible for class switch recombination (CSR) and somatic hypermutation (SHM), was reported to be overexpressed in B cells exposed to HIV-1. Study of primary human B cells in an in-vitro model. In the current study, we evaluated which signalling pathways are activated in primary B cells after a direct contact with HIV-1 particles in vitro using different kinase inhibitors. Here, we report that B-cell activation together with the increase of AID mRNA expression and the subsequent class switch recombination (CSR) in HIV-exposed B cells occurred through spleen tyrosine kinase (SYK) and c-Jun N-terminal kinase (JNK) pathways. Therefore, we showed that HIV-1 could directly induce primary B-cell deregulation via SYK/B-cell receptor (BCR) engagement, and that activation was followed by the JNK pathway activation. To our knowledge, these data provide the first evidence that SYK/BCR activation was the first step for B-cell activation and CSR mechanism after HIV-1 stimulation in a T-cell-free context.

Abstract 5075: B-cell expression of AICDA and MME [CALLA, CD10] is predictive of a subsequent non-Hodgkin lymphoma diagnosis

Abstract Background: Mounting evidence from epidemiologic studies of serum biomarkers highlights the importance of inflammation and immune activation preceding the diagnosis of B-cell non-Hodgkin lymphoma (NHL). Although chronic B-cell hyperactivation is likely involved in the pathogenesis of NHL, an important gap in knowledge remains present as the source and targets of these inflammatory and immune activation associated factors are mainly undefined. Targeted studies of B-cell gene expression can help fill this gap, further define the immune environment from which these tumors arise, and assist in identifying biomarkers for early detection of B-cell NHLs. Methods: This study was based in a large national cohort of HIV+ men, the Multicenter AIDS Cohort Study, who are at increased risk for developing NHL. 144 HIV+ B-cell NHL cases were matched to 144 HIV+ controls on follow-up time and sample availability. B-cells isolated from viably frozen peripheral blood mononuclear cells, collected 2 to 13 years (median 4.7 years) prior to NHL diagnosis or control match date, were used to measure messenger RNA (mRNA) levels of 16 molecules that are associated with B-cell activation, using the Quantigene® Plex 2.0 Assay Kit (Affymetrix) on the Luminex platform. Expression levels were normalized by the geomean of five housekeeping genes. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by conditional logistic regression for gene expression categorized as detectable versus undetectable. Results: Activation-induced cytidine deaminase (AICDA) was detected in 8.3% of cases and 2.8% of controls, OR=3.7, 95% CI=1.0-13.1. Membrane metallo-endopeptidase (MME) was detected in 59.7% of cases and 29.9% of controls, OR=3.9, 95% CI=2.2-6.8. The AICDA and MME associations with NHL were similar across subgroups of site, EBV tumor status, and lag time between sample collection and NHL diagnosis date. AICDA, but not MME, was more strongly associated with NHL among those who were the least immunosuppressed (higher CD4+ T cell count and low HIV RNA levels). In subgroup analyses, CD40 ligand (CD40LG) expression was associated with NHL with a time lag time of &amp;lt;6 years, OR=3.0, 95% CI=1.0-9.3, and Interleukin-6 (IL6) expression was associated with NHL among those with a CD4+ T cell count &amp;lt;300, OR=3.9, 95% CI=0.9-17. Interpretation &amp; Conclusions: AICDA expression is essential for antibody specificity and diversity but its aberrant expression has been implicated in lymphomagenesis. MME is a signature marker of B-cell NHLs of germinal center origin and has previously been associated with NHL survival. We found that AICDA and MME expression precedes the clinical diagnosis of NHL in an HIV+ cohort. Additionally, late CD40LG expression and IL6 expression among the more severely immunosuppressed individuals were associated with NHL risk in this study. These molecules should be studied further for possible early detection and clinical utility. Citation Format: Shehnaz K. Hussain, Marta Epeldegui, Larry I. Magpantay, Elizabeth Crabb Breen, Emilee Knowlton, Steven Wolinksy, Lisa P. Jacobson, Jay H. Bream, Roger Detels, Zuo-Feng Zhang, Otoniel Martinez-Maza. B-cell expression of AICDA and MME [CALLA, CD10] is predictive of a subsequent non-Hodgkin lymphoma diagnosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5075. doi:10.1158/1538-7445.AM2014-5075

Abstract 5350: Aberrant activation-induced cytidine deaminase (AID) expression is induced in the process of hepatitis C virus-associated hepatocarcinogenesis

Abstract Activation-induced cytidine deaminase (AID) is a DNA mutator enzyme, which is encoded by Aicda gene. Under physiological condition, AID is essential for somatic hypermutation of immunogloburin genes in activated B lymphocytes and involved in immune diversity. Previous in vitro studies demonstrated that in cultured adult hepatocytes, both the response to chronic inflammation and hepatitis C virus (HCV) infection itself triggers aberrant AID upregulation, and due to the mutagenic activity of AID, nucleotide alterations gradually accumulate in tumor-related genes. In this study, we aimed to obtain a direct evidence of AID expression induced by HCV infection in vivo using human and mice subjects. Immunostaining of human clinical specimen revealed endogenous AID expression was detectable in both the hepatocellular carcinoma (HCC) and the non-tumorous inflamed liver tissues in patients with chronic HCV infection while no AID expression was detected in normal liver tissues without HCV infection. In mice, as endogenous AID expression level of liver is not sufficient to detect by immustaining, we utilized the transgenic mouse (Aicda-cre transgenic mouse), which was generated by inserting the coding sequence of Cre recombinase into the locus of the Aicda gene, crossing with a genetically-marked reporter mouse (Rosa-tdRFP mouse). In this Aicda-cre/Rosa-tdRFP mouse model, AID-expressing cells can be detected as RFP-positive. We injected HCV core antigen-inserted plasmid to the liver of Aicda-cre/Rosa-tdRFP mice by “hydrodynamic tail vein injection” procedure. 14days after injection, RFP-positive hepatocytes were detected in the HCV antibody-labelled liver cells whereas no RFP-positive cells were found in control mice with physiological saline injection, indicating HCV core antigen induced aberrant AID expression in hepatocytes. Next, to examine the effect of constitutive AID expression in hepatic-lineage cells, we established a transplantation model of hepatic progenitor cells derived from AID Tg mice. We enriched hepatic progenitor cells from the E13.5 fetal liver of AID Tg mice and transplanted those cells into recipient mice. HCC developed in 7 of 11 (63.6%) recipient mice receiving enriched hepatic progenitor cells from AID Tg mice. On the other hand, no tumor was found in recipient mice transplanted with hepatic progenitor cells derived from wild-type mice (control). In addition, whole exome sequencing of tumor cells clarified the landscape of the accumulated genetic alterations during tumorigenesis and detected a large number of somatic mutations in each tumor tissue originating from the transplanted hepatic progenitor cells of AID Tg mice. These results suggested aberrant AID production induced by HCV infection could contribute to hepatocarcinogenesis via the accumulation of genetic alterations by its mutagenic activity. Citation Format: Soo Ki Kim, Hiroyuki Marusawa, Tsutomu Chiba. Aberrant activation-induced cytidine deaminase (AID) expression is induced in the process of hepatitis C virus-associated hepatocarcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5350. doi:10.1158/1538-7445.AM2014-5350

Abstract 5366: Mutation signature of TP53 gene in H. pylori-associated inflamed gastric mucosa during gastric carcinogenesis

Abstract Helicobacter pylori (H. pylori) infection and the resultant chronic gastric inflammation is an important etiologic factor for gastric cancer development. To explore the genetic basis of gastric cancer that develops in inflamed gastric mucosa, we investigated genetic aberrations that latently accumulate in non-tumorous gastric epithelium with H. pylori infection. Whole exome sequencing on gastric cancer, non-cancerous gastritis tissues and peripheral lymphocytes of 5 patients revealed that somatic mutations accumulated in various genes in inflamed gastric tissues. Additional deep sequencing analyses of selected tumor-related genes in 34 gastritis mucosa samples from patients with, or without, gastric cancer revealed non-synonymous low-abundance mutations in TP53 in 15 cases (44.1%) and ARID1A in 5 cases (14.7%). The mutation signature of whole exome sequences in gastric cancer genomes as well as low-abundance TP53 mutations latently accumulated in H. pylori-inflamed gastritis mucosa were predominantly biased to C:G&amp;gt;T:A transitions in the context of GpCpX motifs, the typical footprints of cytidine deamination triggered by activation-induced cytidine deaminase (AID). In vivo studies with a human TP53 knock-in mouse model revealed that constitutive AID expression in the gastric mucosa caused the emergence of human TP53 mutations at the amino acid positions identical to those detected in human gastric cancers. These findings provide novel evidence that somatic mutations latently accumulated in various genes in inflamed gastric mucosa with H. pylori infection, and cytidine deaminase activity might provide a putative genetic basis for the increased susceptibility to gastric carcinogenesis in patients with H. pylori infection. Citation Format: Takahiro Shimizu, Hiroyuki Marusawa, Tsutomu Chiba. Mutation signature of TP53 gene in H. pylori-associated inflamed gastric mucosa during gastric carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2014-5366

Local Immunoglobulin Class Switch Recombination in Chronic Rhinosinusitis with Nasal Polyposis

Objectives: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is characterized by B cell inflammation. We sought to determine whether immunoglobulin class switch recombination (CSR) occurred locally within polyps and to quantify the expression of proteins involved in regulating CSR. Germline transcripts (GLTs) are produced during CSR, and activation-induced cytidine deaminase (AICD) is an enzyme that is required for CSR. Thus, GLT and AICD expression can be used as markers for CSR. Methods: Polyp (n = 12-37), tonsil (n = 7), and uncinate tissue (UT) from patients with CRSwNP (n = 9), CRSsNP (n = 10), and normal controls (n = 11-12) were collected. Quantitative reverse transcriptase polymerase chain reaction for GAPDH, GUSB (housekeeping genes), AICD, IL-15, iNOS, BAFF, and A1, A2, G1, G4, and E GLT was performed using cDNA from tissue. Relative expression was calculated in relation to a housekeeping gene. Results: AICD expression was significantly increased in tonsils compared with all other groups ( P &lt; .05), and polyp AICD expression was significantly elevated compared with UTwNP and UTsNP ( P &lt; .01). Tonsils expressed significantly more A2 GLT than all other groups ( P &lt; .05). E GLT was significantly increased in polyps versus UTnl ( P &lt; .05). G1 GLT was increased in polyps compared with all other groups ( P &lt; .05 vs UTwNP). G4 GLT was also increased in polyps compared with all other tissues, but this did not reach significance. Only IL-15 showed significantly increased expression in polyps versus UTnl and UTwNP ( P &lt; .01). Conclusions: These results suggest that local CSR is occurring in polyps of patients with CRSwNP.

AID expression in peripheral blood of children living in a malaria holoendemic region is associated with changes in B cell subsets and Epstein-Barr virus.

The development of endemic Burkitt's lymphoma (eBL) is closely associated with Epstein-Barr virus (EBV) infection and holoendemic malaria infections. The role of EBV in the development of malignancy has been studied in depth, but there is still little known about the mechanisms by which malaria affects Burkitt's lymphomagenesis. Activation induced cytidine deaminase (AID) expression is necessary for the introduction of c-myc translocations that are characteristic of BL, but a link between AID and EBV or malaria is unclear. To determine whether frequency of malaria exposure leads to increased AID expression in peripheral blood mononuclear cells (PBMC) we examined two cohorts of children in western Kenya with endemic and sporadic malaria transmission dynamics. High frequency of malaria exposure led to increased expression of AID, which coincided with decreases in the IgM(+) memory B cells. In the children from the malaria endemic region, the presence of a detectible EBV viral load was associated with higher AID expression compared to children with undetectable EBV, but this effect was not seen in children with sporadic exposure to malaria. This study demonstrates that intensity of malaria transmission correlates with AID expression levels in the presence of EBV suggesting that malaria and EBV infection have a synergistic effect on the development of c-myc translocations and BL.

NEDD4 ubiquitinates TRAF3 to promote CD40-mediated AKT activation.

CD40, a member of tumour necrosis factor receptor (TNFR) superfamily, has a pivotal role in B-cell-mediated immunity through various effector pathways including AKT kinase, but the signal transduction of CD40-meidated AKT activation is poorly understood. Here we report that the neural precursor cell expressed developmentally downregulated protein 4 (NEDD4), homologous to E6-AP Carboxyl Terminus family E3 ubiquitin ligase, is a novel component of the CD40 signalling complex. It has a key role in CD40-mediated AKT activation and is involved in modulating immunoglobulin class switch through regulating the expression of activation-induced cytidine deaminase. NEDD4 constitutively interacts with CD40 and mediates K63-linked ubiquitination of TNFR-associated factor3 (TRAF3). The ubiquitination of TRAF3 by NEDD4 is critical for CD40-mediated AKT activation. Thus, NEDD4 is a previously unknown component of the CD40 signalling complex necessary for AKT activation.

Intrathecal somatic hypermutation of IgM in multiple sclerosis and neuroinflammation

Intrathecal oligoclonal bands of the cerebrospinal fluid are considered the most important immunological biomarkers of multiple sclerosis. They typically consist of clonally expanded IgG antibodies that underwent affinity maturation during sustained stimulation by largely unknown antigens. In addition, ∼40% of patients with multiple sclerosis have oligoclonal bands that consist of expanded IgM antibodies. We investigated the molecular composition of IgM- and IgG-chains from cerebrospinal fluid of 12 patients with multiple sclerosis, seven patients with other neurological diseases, and eight healthy control subjects by high-throughput deep-sequencing and single-cell PCR. Further, we studied the expression of activation-induced cytidine deaminase, the key enzyme for affinity maturation of antibodies, in cerebrospinal fluid samples of 16 patients. From the cerebrospinal fluid of two multiple sclerosis patients we isolated single B cells and investigated the co-expression of antibody chains with activation-induced cytidine deaminase. In striking contrast to IgM-chains from peripheral blood, IgM-chains from cerebrospinal fluid of patients with multiple sclerosis or neuroborreliosis showed a high degree of somatic hypermutation. We found a high content of mutations that caused amino acid exchanges as compared to silent mutations. In addition, more mutations were found in the complementarity determining regions of the IgM-chains, which interact with yet unknown antigens, as compared to framework regions. Both observations provide evidence for antigen-driven affinity maturation. Furthermore, single B cells from the cerebrospinal fluid of patients with multiple sclerosis co-expressed somatically hypermutated IgM-chains and activation-induced cytidine deaminase, an enzyme that is crucial for somatic hypermutation and class switch recombination of antibodies and is normally expressed during activation of B cells in germinal centres. Clonal tracking of particular IgM(+) B cells allowed us to relate unmutated ancestor clones in blood to hypermutated offspring clones in CSF. Unexpectedly, however, we found no evidence for intrathecal isotype switching from IgM to IgG. Our data suggest that the intrathecal milieu sustains a germinal centre-like reaction with clonal expansion and extensive accumulation of somatic hypermutation in IgM-producing B cells.

A multifactorial role for P. falciparum malaria in endemic Burkitt's lymphoma pathogenesis.

Endemic Burkitt's lymphoma (eBL) arises from the germinal center (GC). It is a common tumor of young children in tropical Africa and its occurrence is closely linked geographically with the incidence of P. falciparum malaria. This association was noted more than 50 years ago. Since then we have learned that eBL contains the oncogenic herpes virus Epstein-Barr virus (EBV) and a defining translocation that activates the c-myc oncogene. However the link to malaria has never been explained. Here we provide evidence for a mechanism arising in the GC to explain this association. Accumulated evidence suggests that eBL arises in the GC when deregulated expression of AID (Activation-induced cytidine deaminase) causes a c-myc translocation in a cell latently infected with Epstein-Barr virus (EBV). Here we show that P. falciparum targets GC B cells via multiple pathways to increase the risk of eBL. 1. It causes deregulated expression of AID, thereby increasing the risk of a c-myc translocation. 2. It increases the number of B cells transiting the GC. 3. It dramatically increases the frequency of these cells that are infected with EBV and therefore protected from c-myc induced apoptosis. We propose that these activities combine synergistically to dramatically increase the incidence of eBL in individuals infected with malaria.

Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.

A new pathway for central B cell tolerance: interaction of AID, MyD88, and BCR (IRC3P.467)

Abstract Expression of activation-induced cytidine deaminase (AID) in immature and transitional B cells in mice and humans is genetically linked to the first tolerance checkpoint. In the absence of AID, autoreactive immature/transitional-1 (T1) B cells are inefficiently purged and exhibit increased resistant to receptor-induced apoptosis. These substantial effects are surprising given that AID expression in the immature/T1 B cells is only 3% of that in germinal center B cells. We now show that in immature/T1 B cells, but not mature B cells, B-cell antigen receptor and Toll-like receptor (TLR) signaling synergize to elicit high levels of AID expression. This synergy is restricted to intracellular TLR ligands, requires the phospholipase D-dependent compartmentalization of intracellular TLR and activation of the TLR through acidification, and acts to ensure self-tolerance through a process that requires Myd88. In a 3H9 mouse model, we show that suppression of intracellular acidification by administration of chloroquine increased the generation of immature/T1 B cells in proportion to small pre-B cells, suggesting that chloroquine treatment support the survival of autoreactive 3H9 immature/T1 B cells in bone marrow. These findings identify a novel mechanism for central B-cell tolerance and resolve several weaknesses of current models for the first tolerance checkpoint. We suggest that this AID mediated pathway may be the primary mechanism for central B-cell tolerance by deletion.