Specific Expression of Activation-induced Cytidine Deaminase (AID), a Novel Member of the RNA-editing Deaminase Family in Germinal Center B Cells

Abstract Several epidemiologic studies have demonstrated an increased risk of non-Hodgkin B cell lymphoma with exposure to pesticides, including herbicides, fungicides and insecticides. Few studies have specifically demonstrated an increased risk for follicular lymphoma in individuals exposed to pesticides. The molecular mechanisms of these associations have not been fully investigated. Activation-induced cytidine deaminase (AID), which physiologically mediates somatic hypermutation in germinal center B cells, has been found to have off-target effects that play an important role in oncogenic mutagenesis driving germinal center type B cell lymphomas. Elevated levels of AID among peripheral blood mononuclear cells has been found to be associated with higher numbers of circulating follicular lymphoma-like cells in individuals exposed to pesticides. Increased AID expression and/or activity represents a possible mechanistic link between pesticide exposure and lymphomagenesis. In order to test the hypothesis that pesticides might lead to an increased risk of germinal center neoplasia by increasing AID levels, four pesticides including isoproturon (a phenylurea herbicide), chlorpyrifos (an organophosphate insecticide), DDT (an organochlorine insecticide), and lindane (an organochlorine insecticide), were screened for their ability to increase AID expression in germinal center type B cells (Ramos and Raji, Burkitt lymphoma cell lines). After 48 hours of treatment with concentrations ranging from 50-200 μM, RNA was extracted, reverse transcribed and cDNA subject to SYBR green Realtime PCR amplification of AID. AID was found to be expressed, at low levels, in untreated Ramos and Raji cells, compared to acute myeloid leukemia cell lines HL60 and U937, which served as negative controls showing no AID expression. Isoproturon treatment resulted in a dose dependent, up to 6-fold, increase in AID in Ramos cells. A smaller, up to 1.9 fold increase was seen in Raji cells. None of the other pesticide treatments resulted in increased expression of AID (Fig. 1). The findings demonstrate that AID expression is induced by isoproturon, a member of the class of phenylurea herbicides which is among the most commonly used herbicides worldwide and generally considered to be relatively safe for humans. These results support a possible role for off-target AID activity in pesticide-exposure related lymphomagenesis. Future studies will investigate the mechanism by which AID is upregulated andwhether or not there is induction of oncogenic mutagenesis in germinal center B cells exposed to phenylurea herbicides. Disclosures No relevant conflicts of interest to declare.

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Activation Induced Cytidine Deaminase (AID) Is Required for Germinal-Center Derived Lymphomagenesis.

Blood ◽

10.1182/blood.v108.11.223.223 ◽

2006 ◽

Vol 108 (11) ◽

pp. 223-223

Author(s):

Laura Pasqualucci ◽

Mara Compagno ◽

Tongwei Mo ◽

Paula Smith ◽

Herbert C. Morse ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Somatic Hypermutation ◽

Profile Analysis ◽

Cytidine Deaminase ◽

Receptor Gene ◽

Activation Induced Cytidine Deaminase ◽

Hodgkin's Lymphomas

Abstract Most B cell non-Hodgkin’s lymphomas (B-NHL) derive from germinal center (GC) B cells and their pathogenesis is associated with the accumulation of distinct genetic lesions, including chromosomal translocations and a more recently identified mechanism of genomic instability, termed aberrant somatic hypermutation. These alterations are thought to be due to mistakes occurring during two GC-associated immunoglobulin (Ig) genes remodeling processes: class switch recombination (CSR) and somatic hypermutation (SHM). However, this model has never been formally proven. To conclusively investigate the role of CSR and SHM in the pathogenesis of B-NHL, we examined whether lymphoma development in mice requires the function of activation induced cytidine deaminase (AID), a DNA editing enzyme expressed specifically in GC and activated B cells and essential for both processes. Three transgenic mouse models were generated by crossing lymphoma-prone mice (λMYC, λMYC/IμHABCL6 and IμHABCL6) with mice (AID−/−) that are unable to undergo both SHM and CSR. The λMYC mice develop a diffusely infiltrating monoclonal proliferation of pre-GC origin, with unmutated IgV genes and lack of BCL6 expression, and therefore presumably independent from AID-associated DNA remodeling events. Conversely, lymphomas in λMYC/IμHABCL6 and IμHABCL6 mice recapitulate GC/post GC-derived malignancies, in that the former display somatically mutated IgV genes and upregulation of post-GC markers (CD138) in most of the cases, while the latter develop a splenic lymphoproliferative syndrome that culminates, past 12 months of age, in clonal B cell lymphomas with DLBCL morphology and somatically mutated IgV genes (~70% of the animals) (Cattoretti et al., Cancer Cell 7:445–455, 2005). Mice were monitored for tumor incidence and survival, and a combination of histologic, immunophenotypic and gene expression profiling analysis was used for tumor characterization. As expected, no significant differences in event-free survival and lymphoma type were observed between AID-proficient and AID-deficient λMYC mice, in agreement with their pre-GC derivation. Conversely, a phenotypic shift of the tumor was observed in λMYC/IμHABCL6 mice when bred into an AID−/− background, with >80% of the cases (N=21/26) reverting to a pre-GC phenotype (loss of GC/post GC markers) undistinguishable from that of the λMYC and λMYC/AID−/− mice. Gene expression profile analysis on representative cases (N=10 λMYC/IμHABCL6 and 5 each for λMYC, λMYC/AIDKO, λMYC/IμHABCL6/AIDKO) confirmed significant phenotypic similarities between pre-GC derived λMYC lymphomas and the λMYC/IμHABCL6/AID −/− lymphomas, which co-segregated in a separate cluster from λMYC/IμHABCL6 tumors. Analogously, a significant reduction in DLBCL frequency was observed in the IμHABCL6/AIDKO cohort as compared to IμHABCL6 mice (N= 4/19, 21% vs 8/14, 57%; p=0.03). Taken together, these results indicate that GC-derived lymphomas cannot develop in the absence of AID, thereby providing direct support to the notion that AID-mediated mistakes in antigen receptor gene modification events (CSR and SHM) represent major contributors to B-NHL pathogenesis.

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Expression of a Dysfunctional Activation Induced Cytidine Deaminase Is Correlated with Disease Progression in Splenic Marginal Zone Lymphoma.

Blood ◽

10.1182/blood.v120.21.2397.2397 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2397-2397

Author(s):

Gabriel Brisou ◽

Laurent Jallades ◽

Alexandra Traverse-Glehen ◽

Francoise Berger ◽

Aurélie Verney ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Germinal Center ◽

Marginal Zone ◽

Cytidine Deaminase ◽

Marginal Zone Lymphoma ◽

Splenic Marginal Zone Lymphoma ◽

Marginal Zone B Cells ◽

Activation Induced Cytidine Deaminase ◽

Splicing Variants

Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.

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AID constrains germinal center size by rendering B cells susceptible to apoptosis

Blood ◽

10.1182/blood-2009-03-211763 ◽

2009 ◽

Vol 114 (3) ◽

pp. 547-554 ◽

Cited By ~ 65

Author(s):

Ahmad Zaheen ◽

Bryant Boulianne ◽

Jahan-Yar Parsa ◽

Shaliny Ramachandran ◽

Jennifer L. Gommerman ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Cytidine Deaminase ◽

Enzyme Activation ◽

Specific Enzyme ◽

Tissue Microenvironment ◽

Activation Induced Cytidine Deaminase ◽

Immunoglobulin Locus

Abstract The germinal center (GC) is a transient lymphoid tissue microenvironment that fosters T cell–dependent humoral immunity. Within the GC, the B cell–specific enzyme, activation-induced cytidine deaminase (AID), mutates the immunoglobulin locus, thereby altering binding affinity for antigen. In the absence of AID, larger GC structures are observed in both humans and mice, but the reason for this phenomenon is unclear. Because significant apoptosis occurs within the GC niche to cull cells that have acquired nonproductive mutations, we have examined whether a defect in apoptosis could account for the larger GC structures in the absence of AID. In this report, we reveal significantly reduced death of B cells in AID−/− mice as well as in B cells derived from AID−/− bone marrow in mixed bone marrow chimeric mice. Furthermore, AID-expressing B cells show decreased proliferation and survival compared with AID−/− B cells, indicating an AID-mediated effect on cellular viability. The GC is an etiologic site for B-cell autoimmunity and lymphomagenesis, both of which have been linked to aberrant AID activity. We report a link between AID-induced DNA damage and B-cell apoptosis that has implications for the development of B-cell disorders.

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CD45RO enriches for activated, highly mutated human germinal center B cells

Blood ◽

10.1182/blood-2007-05-087767 ◽

2007 ◽

Vol 110 (12) ◽

pp. 3917-3925 ◽

Cited By ~ 11

Author(s):

Stephen M. Jackson ◽

Natessa Harp ◽

Darshna Patel ◽

Jeffrey Zhang ◽

Savannah Willson ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Somatic Mutation ◽

Germinal Center ◽

Cytidine Deaminase ◽

Annexin V ◽

Cell Development ◽

B Cell Development ◽

Activation Induced Cytidine Deaminase

Abstract To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker.

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MicroRNA 155 Control of p53 Activity Is Context Dependent and Mediated by Aicda and Socs1

Molecular and Cellular Biology ◽

10.1128/mcb.01446-14 ◽

2015 ◽

Vol 35 (8) ◽

pp. 1329-1340 ◽

Cited By ~ 24

Author(s):

Hakim Bouamar ◽

Daifeng Jiang ◽

Long Wang ◽

An-Ping Lin ◽

Manoela Ortega ◽

...

Keyword(s):

B Cells ◽

Germinal Center ◽

Cytidine Deaminase ◽

Biological Processes ◽

Functional Interactions ◽

Activation Induced Cytidine Deaminase ◽

Damage Response ◽

P53 Response ◽

P53 Activation

In biological processes, the balance between positive and negative inputs is critical for an effective physiological response and to prevent disease. A case in point is the germinal center (GC) reaction, wherein high mutational and proliferation rates are accompanied by an obligatory suppression of the DNA repair machinery. Understandably, when the GC reaction goes awry, loss of immune cells or lymphoid cancer ensues. Here, we detail the functional interactions that make microRNA 155 (miR-155) a key part of this process. Upon antigen exposure, miR-155−/−mature B cells displayed significantly higher double-strand DNA break (DSB) accumulation and p53 activation than their miR-155+/+counterparts. Using B cell-specific knockdown strategies, we confirmed the role of the miR-155 target Aicda (activation-induced cytidine deaminase) in this process and, in combination with a gain-of-function model, unveiled a previously unappreciated role for Socs1 in directly modulating p53 activity and the DNA damage response in B lymphocytes. Thus, miR-155 controls the outcome of the GC reaction by modulating its initiation (Aicda) and termination (Socs1/p53 response), suggesting a mechanism to explain the quantitative defect in germinal center B cells found in mice lacking or overexpressing this miRNA.

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