Activation-induced Cytidine Deaminase mRNA Research Articles

Abstract A49: Induction of the lymphomagenic enzyme activation-induced cytidine deaminase (AID) upon in vitro exposure to phenylurea herbicides

Abstract Many studies have drawn associations between pesticide exposures and lymphomagenesis, both in occupational settings and in the general population. The specific compounds responsible for an increased risk of non-Hodgkin lymphoma in pesticide-exposed individuals have not been entirely established. Furthermore, the biologic mechanisms by which pesticides act as lymphomagens are unknown. One mechanism of B-cell genomic instability that plays a role in lymphomas derived from germinal center (GC)-type B cells, including follicular lymphoma, diffuse large B cell lymphoma, and Burkitt lymphoma, is the off-target/aberrant activity of activation-induced cytidine deaminase (AID). AID damages DNA through deamination of cytidine. This occurs on a physiologic basis within GC B cells and is responsible for both class switch recombination (CSR) and somatic hypermutation (SHM). AID can also target nonimmunoglobulin loci, including several proto-oncogenes, resulting in both mutations (aberrant somatic hypermutation; aSHM) and chromosomal translocations. Overexpression of AID has previously been found to increase B-cell genomic instability. In this study, GC-type B cell lines (Ramos, Raji, and CL-01) were treated with pesticides, including herbicides and insecticides from various classes, to determine the effect on AID expression. Real-time RT-PCR was used to determine the degree of AID mRNA expression after chemical treatment. Two representative phenylurea herbicides, linuron and diuron, were found to induce AID expression, in a time- and dose-dependent manner, at concentrations ranging from 50-200 micromolar, with maximal induction of 2- to 6-fold depending on the compound and time point. Additional pesticides that were tested but not found to induce AID expression, at concentrations that were minimally toxic in vitro, included representative organochlorine, organophosphate, phenoxy, and phosphanoglycine pesticides. The findings raise the possibility of increased AID-mediated genomic instability in GC type B cells exposed to phenylurea herbicides. Future studies will investigate the possible link between the observed phenylurea herbicide-related induction of AID, B-cell genomic instability, and lymphomagenesis. Citation Format: Rebecca J. Leeman-Neill, Uttiya Basu. Induction of the lymphomagenic enzyme activation-induced cytidine deaminase (AID) upon in vitro exposure to phenylurea herbicides [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A49.

FHL2 Is Essential for Spleen T Cell-Dependent B Cell Activation and Antibody Response.

Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule regulating various cellular processes, including signal transduction, transcription, and cell survival. Although involved in inflammation and immune responses, its role in the germinal center reaction and B cell maturation remains unknown. We found that FHL2-/- mouse spleens displayed enlarged follicles with more B cells. When a T cell-dependent immune response was elicited using SRBC, FHL2-/- germinal center area was enhanced 2-fold compared with wild type (WT), concomitant with expanded dark zones. Nevertheless, the SRBC-induced rise in spleen IgG1 expression, and plasma IgG1 levels observed in WT were absent in FHL2-/- mice, and circulating plasma cells were also reduced in FHL2-/- This could be explained by deficient upregulation of spleen activation-induced cytidine deaminase mRNA. Interestingly, FHL2-/- B cells successfully underwent class-switch recombination in vitro, and both activation-induced cytidine deaminase induction and IgG1 response to SRBC were equivalent in B cell-deficient μMT mice transplanted with WT or FHL2-/- bone marrow, suggesting that the defects observed in FHL2-/- mice were not B cell intrinsic. However, spleen lysates from FHL2-/- mice revealed a disturbed spleen microenvironment, with reduced CXCL12 and CXCL13 levels compared with WT. Our data suggest that spleen FHL2 expression is essential for a normal germinal center reaction and proper induction of class-switch recombination in response to a T cell-dependent Ag, leading to the emergence of Ab producing plasma cells. This could be due to the regulation of spleen cytokine production by FHL2.

Intraclonal Diversification Occurs in Chronic Lymphocytic Leukemia Expressing B Cell Receptors Belonging to the IGHV4 Gene Family

Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era.Aim. To apply a NGS approach to investigate ID in CLL.Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS <4) and cases with ID (intraclonal; DS ≥4) (Fig. 1B).Results. Using the reported threshold we identified 469 (88.5%) clonal cases, expressing a single clone (Fig. 1C), and 61 (11.5%) cases with ID (median DS 9.2, range 4.4-66.0) characterized by the presence of two or multiple pathological clones expressing the same IGHV gene and HCDR3 (Fig. 1C). Notably, cases with ID expressed both a mutated (M) (39/61, 63.9%) and an unmutated (UM) (22/61, 36.1%; p=0.066) IGHV gene configuration (Fig. 1C). Of note, we observed a significant skewing toward the usage of VH4-family genes when comparing cases with ID (38/61, 62.3%) vs. cases without ID (78/469, 16.6%; p<0.0001, Fig 1D). Moreover, the IGHV4-39 and IGHV4-34 genes were the most used genes in the context of cases with ID (Fig. 1E), although none of them belonging to known stereotyped subsets. By focusing on VH4-family only cases, we observed that cases with ID and UM IGHV genes displayed higher mutation frequencies in WA/TW motifs, a mutational signature which suggests an involvement of both Activation-Induced (Cytidine) Deaminase (AID) and error-prone polymerase eta (Fig. 1F), a pattern not observed in its counterpart with UM IGHV genes but without ID (Fig. 1F). Conversely, in cases with ID and M IGHV genes, mutations preferentially clustered in AID hotspots (WRC/GYW motifs), suggesting a direct role of AID and the Base Excision Repair machinery in the mutational overload (Fig. 1F). Consistently, M IGHV cases with ID expressed significantly higher AID mRNA levels than M IGHV cases without ID (p=0.0024; Fig. 1G). These expression levels were overall comparable with those found in UM IGHV cases, irrespective to the evidence of ID (Fig. 1G), which however were not associated with an increased number of mutations in AID-specific hotspots (Fig. 1F).Conclusions. By taking advantage of a new method for ID assessment in CLL, we demonstrated that ID prevalently affects VH4-family cases which display different mutational patterns dependent to the IGHV gene status. This data are in keeping with previous reports indicating the IGHV4 genes as particularly prone to generate immunoglobulin subjected to continuous/persistent stimulation by external/auto-antigens, hence particularly prone to generate features of ID. Further experiments in selected cases with ID through a non-random barcode strategy are needed. DisclosuresZaja:Sandoz: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Abbvie: Honoraria.

Enhanced B-cell differentiation driven by advanced cirrhosis resulting in hyperglobulinemia.

The mechanism underlying hyperglobulinemia in cirrhosis, a long appreciated phenomenon, has never been clearly understood. The aim of this study is to investigate the basis for changes in humoral immunity observed in cirrhosis. We retrospectively reviewed our medical record to analyze serum immunoglobulin (Ig) levels in patients with liver disease. We also prospectively analyzed peripheral blood mononuclear cells and sera from liver disease patients. Peripheral blood mononuclear cell surface marker expressions were measured by flow cytometry and serum B-cell-activating factor was measured by enzyme-linked immunosorbent assay. Expression of specific gene expression in magnetically separated B cells was also analyzed by real-time polymerase chain reaction. In retrospective analysis, we found that advancing cirrhosis, irrespective of underlying etiology or hepatocellular carcinoma, resulted in progressively increasing levels of serum IgG and IgA. In prospective analysis using clinical samples, we demonstrated that advancing cirrhosis stage was associated with increased toll-like-receptor (TLR)9 expression in CD27+ B cell and serum B-cell-activating factor levels but decreased CD27+ memory B-cell frequency. The remaining CD27+ B cells in peripheral blood exhibited increased activation-induced cytidine deaminase mRNA expression. Finally, we also demonstrated isolated B cells from advanced cirrhosis were more reactive to TLR9 stimulation that drove antibody secreting cells differentiation leading to hyperimmunoglobulinemia in vitro. Enhanced TLR9-induced differentiation into antibody secreting cell may explain peripheral reductions of circulating CD27+ memory B cells as well as increased serum Ig levels in cirrhosis.

High expression of activation-induced cytidine deaminase mRNA can predict early-stage progression and poor responsiveness to treatment in chronic lymphocytic leukemia

High expression of activation-induced cytidine deaminase mRNA can predict early-stage progression and poor responsiveness to treatment in chronic lymphocytic leukemia

Abstract B31: The role of HIV protein Nef in the development of HIV/AIDS-associated lymphomas

Abstract Introduction: The pathogenesis HIV-associated lymphomas involve a complex interplay of biological factors, including the immerging role of HIV encoded proteins. These proteins have been shown to have oncogenic potential and to enhance the pathogenesis of HIV-associated malignancies. However, very little is known about the molecular mechanisms involved in this interaction. Here, we investigate the effect of HIV protein Negative Factor (Nef) on the expression of c-MYC and activation-induced cytidine deaminase (AID) in lymphoblasts and lymphoma cells. Methods: Native histidine-tagged HIV Nef was produced in a bacterial system and used to treat (1) an EBV-immortalized B lymphoblastoid cell line (L1439A) and (2) the established lymphoma cell line Ramos, at various concentrations and for various time points. Changes in the expressions of c-MYC and AID were measured at both the mRNA and proteins levels using qPCR and western blotting respectively. The ability of Nef to regulate the promoter of these two genes was determined using luciferase reporter assays. Furthermore, confocal microscopy was used to investigate the downstream effects of Nef exposure. Results: In L1439A cells, a general decrease in the expression of c-MYC and AID was observed. After 1 and 3 hours of exposure to HIV-Nef both c-MYC and AID mRNA levels reduced by approximately 0.5-fold. In contrast, the expressions of both genes increased significantly upon these treatments in the lymphoma cell line Ramos. After 3 hours of exposure, c-MYC expression increased by ~1.9-fold, while AID expression increased up to 4.6-fold. The protein expression followed a similar pattern, as indicated by western blotting. Furthermore, luciferase reporter assays showed that the effect on c-MYC expression is likely to be direct since the activity of the c-MYC promoter increased in a dose-dependent manner in the presence of Nef. c-MYC translocated to the nucleus, which is indicative of an active protein. Furthermore, an increase in gamma H2AX foci on DNA, indicative of double strand breaks, shows that the increase in AID lead to genomic instability. Conclusion: This study demonstrates a differential effect of HIV Nef on normal immortalized lymphoblasts versus lymphoma cells. More importantly, it shows for the first time that HIV Nef may enhance the pathogenesis of lymphomas by triggering pathways and mechanisms which increase the expression of c-MYC and AID. Citation Format: Shaheen Mowla, Nontlantla Mdletshe. The role of HIV protein Nef in the development of HIV/AIDS-associated lymphomas [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B31.

Aberrant expression of interleukin-10 and activation-induced cytidine deaminase in B cells from patients with Behçet's disease.

Despite extensive studies, the pathogenesis of Behçet's disease (BD) remains unclear. In particular, the roles of B cells in patients with BD have not been elucidated. Activation-induced cytidine deaminase (AID) is a critical enzyme for immunoglobulin (Ig) heavy chain class switching and somatic hypermutation in B cells and the abnormal expression of AID in various immune conditions has previously been studied. B10 cells, an interleukin (IL)-10-secreting subset of regulatory B cells, function to downregulate inflammation and autoimmunity. Thus, in the present study, the relevance of B cells in patients with BD was investigated. The plasma levels of IL-10 and IgA and the proportions of cluster of differentiation (CD)43+ B cells, excluding naïve B cells, were measured in 16 patients with BD and 16 age- and sex-matched healthy controls (HCs). Additionally, the mRNA levels of IL-10 and AID were assessed in B cells from fresh peripheral blood samples of the BD patients and HCs. The plasma level of IL-10 in patients with BD did not differ significantly from that in HCs. Similarly, there was no significant difference in the plasma level of IgA, although a slight increase was observed in patients with BD compared with that in HCs. There were no differences in CD43+CD19+ B cell numbers between patients with BD and HCs. However, IL-10 mRNA levels were significantly reduced (P<0.05), while AID mRNA levels were significantly increased (P<0.01) in the B cells of patients with BD compared with those in HCs. These results provide insight into the role of B cells in patients with BD.

Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells.

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.

PI3Kdelta Inhibitors Increase Genomic Instability By Upregulating Aid Expression

▪Activation-induced cytidine deaminase (AID) is a B cell-specific enzyme that initiates class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes, essential mechanisms to generate different classes of antibody and antibody diversity for the antigens. At lower frequency, AID also promiscuously introduces DNA structural lesions at non-Ig loci and it is involved in the pathogenesis of B cell lymphoma. Thus, its expression is tightly controlled in B cells to limit its genotoxic effects. Phosphatidylinositol 3-kinase (PI3K) p110δ isoform acts downstream of the B-Cell Receptor (BCR) to suppress AID expression, whereas blockade of PI3K signaling enhances the expression of AID. Potent oral PI3K inhibitors such as the p110δ inhibitor idelalisib (GS-1101, CAL-101) have been recently approved for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Waldenstrom macroglobulinemia (WM), whereas dual PI3K p110δ and γ inhibitors such as duvelisib (IPI-145) showed promising results for the treatment of CLL and other hematologic malignancies.Here we investigated whether the increased of AID expression caused by idelalisib or duvelisib induced genomic instability in normal and malignant B cells.We observed that in splenic purified mouse B cells activated with IL4 and αCD40 to induce AID expression and CSR, treatment with idelalisib and duvelisib significantly increased both AID protein and mRNA levels, compared to controls. As a result, both drugs strongly promoted CSR. The regulation of AID expression was strictly dependent on PI3K p110δ activity because a selective p110γ inhibitor (AS-604850) did not have any effect. In contrast, B cells expressing a constitutively active PI3Kδ showed suppression of CSR and reduced AID expression compared to inactive PI3Kδ.To gain insights into the degree of genomic instability induced by increased AID expression upon PI3Kδ inhibition, we applied a genome-wide translocation technique we previously developed (High-Throughput Genomic Translocation Sequencing approach, HTGTS) to identify translocation partners from DNA double strand breaks (DSBs) introduced into the c-myc locus (Chiarle et al, Cell 2011). By HTGTS, we isolated thousands of translocations from activated primary mouse B cells distributed widely across the genome. Remarkably, in B cells treated with idelalisib or duvelisib, we identified a significantly higher number of translocations in known AID off-target genes as well as novel hotspots of translocations (48 for idelalisib, 50 for duvelisib). Unbiased genome-wide analysis of translocation formation revealed a consistency in the translocation patterns, with AID target hotspots localized in the TSS region and predominantly grouped within super-enhancers and regulatory clusters. HTGTS analysis performed on activated AID knock-out (AID KO) B cells showed that the vast majority of these translocations were dependent on AID. Hence, our data demonstrate that in normal B cells PI3Kδ inhibitors increase genomic instability by an AID-dependent mechanism.Finally, we observed that both idelalisib and duvelisib increased AID expression in human EBV-immortalized and lymphoma cell lines (MCL and CLL). We adapted the HTGTS technique to human cells by introducing DSBs in the human myc locus by CRISPR/Cas9 technology and sequencing genome-wide the formation of chromosomal translocations. By this approach, we demonstrated that also in human neoplastic B cells the treatment with idelalisib or duvelisib increased the formation of translocations to known AID off targets.In conclusion, we showed that idelalisib or duvelisib increase genomic instability in normal and neoplastic B cells by enhancing AID expression. Since several B cell malignancies imply treatment with these drugs for years, these effects of PI3Kδ inhibitors on the genomic stability of B cells should be carefully taken into account for therapeutic outcomes and protocol design. DisclosuresNo relevant conflicts of interest to declare.

Molecular Evidence of Ongoing Antigen Driven Somatic Hypermutation in the Natural History of Classic and Variant Hairy Cell Leukemia

Classic hairy cell leukemia (HCL) is a clonal chronic B-cell malignancy representing 2% of all leukemias, with neoplastic B lymphocytes in the blood, bone marrow and organs of the classic reticuloendothelial system. HCL variant (HCLv) is less common, typically has inferior prognosis, and usually lacks several HCL-related features including BRAF V600E, CD25, and Annexin A1. The malignant cells in HCL and HCLv patients have one or in unusual cases two different functional immunoglobulin heavy chain rearrangements. Significant variability can be observed in the 3rd complementarity determining region (CDR3), comprising the variable heavy (IGHV), diversity (IGHD), and joining (IGHJ) domains. A role for continued antigen selection in the pathogenesis and evolution of HCL and HCLv has been suggested but limited evidence has been found.We examined HCL and HCLv-derived IGHV genes by massive parallel deep sequencing in 15 HCL and 7 HCLv cases. In all studied samples, a productive monoclonal tumor-derived IGH sequence was detected. Analysis of all reads sharing clonal CDR3 motifs revealed the existence of dominant subclone expansion and multiple subclonal variants. Analysis of phylogenetic trees indicated multiple branching and therefore exposure to multiple rounds of somatic hypermutation (SHM) in the evolution of clonally-related malignant cells. To investigate a possible cause of the high SHM activity and the large number of subclones in HCL and HCLv, we studied the expression of activation induced cytidine deaminase (AID), an enzyme essential for somatic hypermutations, expressed by germinal center (GC) B-cells where SHM occurs. In 5 HCL and 3 HCLv cases we detected a high expression level of AID mRNA, including wild-type and 2 splice variants; in 10 HCL, 4 HCLv, and 3 normal peripheral blood samples, AID expression was not detected using standard end-point PCR conditions. However, more sensitive real-time quantitative PCR detected AID transcripts in virtually all tested HCL and HCLv cases, although the range of transcript levels was large between different cases and varied with individual cases over time.The data obtained for both HCL and HCLv fit a model of tumorigenesis in which the BRAF V600E mutation (or another event in HCLv) initiates neoplastic transformation in a GC B-cell committed to terminal differentiation, but still thereafter still targeted by ongoing SHM DisclosuresNo relevant conflicts of interest to declare.

In vitro evaluation of B cell function using activation-induced cytidine deaminase in HIV-infected patients on antiretroviral therapy (HUM6P.244)

Abstract Background: B cell populations and function depend on the stage of HIV-1 infection. Good virologic control can normalize B cell populations, but data regarding the effect on B cell function is lacking. Method: We investigated B cell populations and intrinsic function among a perinatally and behaviorally infected HIV-1 patients based on virologic control and correlated both. Peripheral blood monocytes were collected from a HIV-negative (HN), behaviorally infected HIV patients with undetectable viral load of &amp;lt;40 copies/mL (BU) and two perinatally infected groups. These groups were categorized by viral load: undetectable group (PU) of &amp;lt;40 copies/mL and a low level viremia (PLLV) of &amp;lt;200 copies/mL. Activation-induced cytidine deaminase mRNA quantification was used after in vitro stimulation with CpG oligodeoxynucleotides to measure intrinsic B cell function. Results: B cell populations were similar in the HN and BU groups, which differed from the PU and PLLV groups. The PU and PLLV groups demonstrated higher percentages of naïve B cells and lower percentages of IgM and switch memory B cells, demonstrating patterns of cellular aging. However, the exhausted B cell populations were not significantly different between groups. Virologic control did not affect B cell function in the infected groups when compared to the uninfected group. Conclusion: Good virologic control, defined as &amp;lt; 200 viral copies/mL, normalizes B cell populations and more importantly intrinsic B cell function.

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.

Glucocorticoid inhibition of activation-induced cytidine deaminase expression in human B lymphocytes

We examined whether glucocorticoids could modulate the expression of activation-induced cytidine deaminase (AICDA), the principal regulator of the processes of immunoglobulin gene somatic hypermutation and class switch recombination in B lymphocytes. Treatment of human B cells with IL-4 and anti-CD40 antibody for 18–20h resulted in induction of expression of AICDA mRNA by over 10-fold. Dexamethasone at 10nM concentration inhibited AICDA induction by an average of 51.8% (p<0.0001). These effects of glucocorticoids were found to be dose dependent in the physiologic range and were reversible by co-treatment with a glucocorticoid receptor antagonist. Human B cell viability and proliferation were unaltered by glucocorticoid treatment. These data demonstrate that physiologic concentrations of glucocorticoids can act on human B lymphocytes through glucocorticoid receptor-mediated mechanisms to diminish the expression of AICDA, a key regulator of humoral immune responses.

Analysis of activation-induced cytidine deaminase mRNA levels in patients with chronic lymphocytic leukemia with different cytogenetic status

Activation induced cytidine deaminase (AID) enzyme, which converts cytosine into uracil and is expressed only by activated B lymphocytes, plays a role in B cells in both the mechanisms of somatic hypermutation (SHM) and class switch recombination (CSR). There are studies showing that AID can cause numerous translocations in different lymphoproliferative diseases. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells in bone marrow and peripheral blood. The predictability and clinical status of B-CLL are difficult to determine. About 30–50% of patients have chromosomal abnormalities. AID, which is thought to create fraction segments for translocations, might also cause deletions in DNA regions of 17p13, 11q22.3, 13q14 and 13q34 that are associated with prognostic implications in patients with CLL. In this study, the AID gene expression in patients with CLL with and without deletions was investigated. When compared to healthy subjects and patients without deletions, increased levels of AID expression in patients with deletions of 17p13, 11q22.3 or 13q14 were found, but not for the 13q34 region. Our results show that AID expression may be associated with deletions in patients with CLL.

Epstein–Barr virus latent membrane protein 1 increases genomic instability through Egr-1-mediated up-regulation of activation-induced cytidine deaminase in B-cell lymphoma

Epstein–Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) is a transmembrane protein essential for EBV-induced immortalization and transformation of B cells. Activation-induced cytidine deaminase (AID) triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Here, we report an intracellular mechanism by which LMP1 contributes to B cell lymphomagenesis via AID expression. In our experiments, LMP1 increased AID mRNA expression and promoter activity. The AID promoter region contains a binding site for Egr-1, a prominent transcription factor that is reported to be up-regulated by LMP1. In promoter activity analysis, Egr-1 enhanced the reporter activity of the wild-type AID promoter, but not that containing a mutated Egr-1 binding site. Egr-1 knockdown abrogated LMP-1-mediated up-regulation of AID promoter reporter activity in EBV-negative BJAB cells and reduced AID promoter reporter activity in EBV-positive SKW6.4 cells. AID induced down-regulation of the nuclear factor-κB (NFκB) inhibitory tumor suppressor Rassf6, suggesting that AID functions as an upstream regulator of the NFκB inhibitory Rassf6. Moreover, Egr-1 expression was associated with an increased number of genomic lesions in genome-wide analysis using single nucleotide polymorphism (SNP) microarray and copy number variation (CNV). Collectively, LMP1 induces AID up-regulation and genomic instability via Egr-1. Increased AID expression may, in turn, promote down-regulation of the NFκB inhibitor, Rassf6, thereby further increasing the survival of genetically destabilized B-cell lymphoma cells.

Diversification of the primary antibody repertoire begins during early follicle development in the rabbit appendix

Rabbits generate a diversified primary antibody repertoire by somatically mutating, in gut-associated lymphoid tissue (GALT), an initial repertoire that is limited by preferential rearrangement of the 3′-most IGVH gene segment. To determine when repertoire diversification begins in GALT, we performed in situ hybridization on neonatal rabbit appendix sections with an activation-induced cytidine deaminase (AID) riboprobe, because AID is required for the mutational processes that diversify the primary antibody repertoire. We first detected AID mRNA expression around 1 week of age, in the basal region of developing follicles. By PCR-amplifying V–D–J genes from AID mRNA+ B cells isolated by laser capture microdissection, we found evidence of somatic hypermutation, and one likely instance of somatic gene conversion. Our results suggest that V–(D)–J gene diversification begins during early postnatal appendix development, in B cells stimulated to enter a proliferative program by signals derived from select intestinal commensals.

TACI mutations and impaired B-cell function in subjects with CVID and healthy heterozygotes

Mutations in the gene coding for the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) are found in 8% to 10% of subjects with common variable immunodeficiency (CVID). Although heterozygous mutations may coincide with immunodeficiency in a few families, most mutation-bearing relatives are not hypogammaglobulinemic. Thus, the role of TACI mutations in producing the immune defect remains unclear. This study examined the expression and function of TACI mutations in healthy heterozygous relatives. We examined the surface and intracellular expression of TACI protein in EBV-transformed B cells of patients and relatives with mutations in 7 families, binding of a proliferation-inducing ligand, and secretion of IgG and IgA by ligand-activated B cells. We tested whether Toll-like receptor 9 agonists increased TACI expression and whether an agonistic anti-TACI antibody could induce activation-induced cytidine deaminase mRNA in those with mutations. Intracellular and extracellular TACI expression was defective for B cells of all subjects with mutations, including subjects with CVID and relatives. Although Toll-like receptor 9 triggering normally up-regulates B-cell TACI expression, this was defective for all subjects with mutations. Triggering TACI by an agonistic antibody showed loss of activation-induced cytidine deaminase mRNA induction in all mutation-bearing B cells. However, ligand-induced IgG and IgA production was normal for healthy relatives but not for subjects with CVID. Thus, B cells of relatives of subjects with CVID who have mutations in TACI but normal immune globulin levels still have detectable in vitro B-cell defects.

Analysis of cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulin

Although various in vitro immunization methods to generate antigen-specific antibodies have been described, a highly effective method that can generate high-affinity immunoglobulins has not yet been reported. Herein, we analyzed a cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulins. We identified a combination of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies) plus lipopolysaccharides (LPS) that stimulates antigen-exposed splenocytes in vitro, followed by induction of the cells phenotypically equivalent to germinal center B cells. We also observed that LPS induced high expression levels of mRNA for activation-induced cytidine deaminase. We stimulated antigen-exposed splenocytes, followed by the accumulation of mutations in immunoglobulin genes. From the immunized splenocytes, hybridoma clones secreting antigen-specific immunoglobulins were obtained.

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

AbstractAbstract 2397B 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.

High Expression of Activation-Induced Cytidine Deaminase and in Vivo Class Switch Recombination in Mantle Cell Lymphoma: Further Support for Antigen Involvement in Lymphomagenesis

AbstractAbstract 1538According to the WHO 2008 Classification, the cellular origin of mantle cell lymphoma (MCL) is traced to a peripheral B cell of the inner mantle zone, mostly of naïve pre-germinal center type. This notion, however, is seriously challenged by both the remarkable restrictions of the immunoglobulin gene repertoire in MCL and, furthermore, by the fact that the great majority of cases exhibit imprints of somatic hypermutation (SHM) in rearranged IGHV genes, ranging from (mostly) minimal to pronounced. These findings support an antigen-driven origin for MCL, at least for a substantial fraction of the entire cohort. Activation-induced cytidine deaminase (AID) is induced in B cells following contact with antigen and is critically implicated in both somatic hypermutation (SHM) and class switch recombination (CSR). Although the available information about AID expression and in vivo CSR in MCL is limited and contradictory, at least some MCL cases have been reported to express AID and undergo ongoing CSR. With this in mind, here we investigated AID-mRNA isoform expression and isotype switch events in a large series of MCL cases and explored possible associations with IGHV gene repertoire and SHM status. Overall, 107 cases were included in the study and tumor-involved diagnostic tissue samples of different types were evaluated, including: fresh-frozen lymph nodes (LN, n=53), peripheral blood (PB, n=42), spleen (n=5), bone marrow biopsies (n=3) and other (n=4). The neoplastic lymphocytic infiltration ranged from 52–98% (median 80%). Thirty-five of 107 cases (32.7%) carried IGHV genes with 100% identity to the germline (GI) whereas the remaining 72 cases bore some imprint of SHM: in particular, 48/107 cases (44.9%) carried IGHV genes with 97–99.9% GI and, finally, 24/107 cases (22.4%) carried IGHV genes with <97% GI. In keeping with the literature, the IGHV gene repertoire of the present cohort was remarkably biased, with the IGHV3–21, IGHV4–34, IGHV3–23 and IGHV1–8 genes accounting for 55.1% of cases. Profiling of AID mRNA expression was performed by RQ-PCR for the full-length AID (AID-FL) as well as the most frequent splice variants, namely AID-ΔE4a (lacking the first 30 nucleotides from exon 4), and AID-ΔE4 (loss of the entire exon 4). AID transcript levels were calculated as the percentage of AID copy number divided by the copy number of the reference transcript (c-ABL). AID-FL transcripts were detected in 104/107 (97%) cases whereas the AID-ΔE4a and AID-ΔE4 splice variants were detected in 72/107 (67.3%) and 107/107 cases (100%), respectively. The median values for AID-FL, AID-ΔE4a and AID-ΔE4 transcripts were 4.45%, 0.133% and 0.918%, respectively. AID transcript levels varied between different cases by up to 5-log for AID-FL transcripts and 4-log for splice variants. Not unexpectedly, the median transcript levels in LN samples were higher (up to 1-log) compared to PB samples. A highly significant (p<0.001) association was noted between medium-to-high AID-FL transcript levels (AID-FL/ABL○1%) and IGHV GI 100%. Given the difference in tissue origin of our samples, we also performed a separate analysis for LN samples only and found that cases with 100% IGHV GI expressed high AID-FL transcript levels (AID-FL/ABL○10%) significantly (p=0.04) more frequently than cases carrying mutated IGHV genes. Isotype switch events were investigated in 41 cases: overall, 4 cases (9.7%), all with GI<100%, carried alternative tumor-derived Cγ (n=1) or Cα (n=3) transcripts. In conclusion, the present analysis documents AID expression in the vast majority of MCL, thus corroborating our previous hypothesis for antigen involvement in MCL ontogeny. Ongoing CSR events appear to be a feature of MCL, further supporting an activated status, at least for subset of cases. Disclosures:No relevant conflicts of interest to declare.