Demethylase Activity of Aid during Germinal Center B Cell Maturation Could Contribute to Lymphomagenesis

Abstract Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors that arise from germinal center B cells (GCBs) and post-GCBs and are noted for their heterogeneity and variable clinical outcomes. Epigenetic modifications like DNA methylation of cytosine nucleotides have emerged as important mechanisms of gene regulation and have been implicated in carcinogenesis. Our previous genome-wide studies in primary samples revealed profound alterations in the cytosine methylation patterning of DLBCLs. We also found that expression of activation-induced deaminase (AID) was significantly associated with the loss of methylation in DLBCL patients and was predominantly identified within computationally predicted AID-binding RGYW motifs. AID is a cytidine deaminase required for class switch recombination and somatic hypermutation (SHM) of immunoglobulin genes in GCBs. The enzymatic machinery that mediates these processes is error-prone and may introduce point-mutations and changes in DNA methylation, resulting in genomic and epigenomic instability. Since AID can also function as a demethylase during embryonic development, we asked whether AID has demethylase activity during transit of B cells through the GCs and if its overexpression can contribute to lymphomagenesis through disrupting DNA methylation. To address this question, we studied the epigenetic function of AID in GCBs and GC-derived lymphomas. We characterized the methylome of naïve B cells (NBs) and GCBs isolated from human tonsils and spleens of immunized mice by enhanced Reduced Representation Bisulfite Sequencing (eRRBS). We observed that the transition from NBs to GCBs was characterized by DNA hypomethylation, with 60,000 and 8,000 differentially methylated CpGs (DMCs) that were hypomethylated in GCBs compared to NBs, in human and mouse respectively. We also found that hypomethylated regions were enriched for the putative AID binding site RGYW (Wilcoxon P <.001). Furthermore, AID knockdown in lymphoma cells (RAMOS) resulted in preferential hypermethylation at AID-binding sites (Chi square P ~ 0). We then isolated DNA from splenic NBs and GCBs from Aicda-/- (AID-deficient) and Aicda+/+ (wild type) mice and performed eRRBS analysis, obtaining single nucleotide resolution for 2.5-3 million represented CpGs. We observed that most of the 8,000 hypoDMCs identified between GCBs and NBs in Aicda+/+ mice were absent in Aicda-/- mice (800 hypoDMCs between GCBs and NBs Aicda-/- cells), implying that AID is a regulator of DNA methylation in GCBs. In addition, those AID-dependent hypoDMCs were predominantly localized in introns (35%), and also in promoters (10%) and exons (10%). We then defined differentially methylated regions (DMRs) based on the following criteria: ≥ 5 DMCs and methylation difference ≥10%, with >250bp between DMRs. We identified DMRs that get hypomethylated in GCBs in the Aicda+/+ mice, but are not hypomethylated in Aicda-/- GCBs, corresponding to >200 genes that represent AID epigenetic targets. These genes include factors involved in B cell function and differentiation like PAX5, BCL2L1, IRF8 and others. Not unexpectedly, many of epigenetic targets are also known targets for SHM, but some are novel targets that only demonstrate evidence of epigenetic deregulation. We also analyzed the transcriptome of NBs and GCBs from Aicda-/- and Aicda+/+ mice by RNA-seq and detected an increase in DNMT1 expression in Aicda-/- cells compared to Aicda+/+ cells. There were no significant changes in expression of other factors involved in modification of cytosine methylation, such as DNMT3a/3b, TET1/2/3, UNG or MSH2/6. Finally, we performed bone marrow transplantation experiments using VavP-Bcl2 mice, which are known to develop GC-derived lymphomas. We transplanted VavP-Bcl2 bone marrow cells infected with AID-expressing retroviral vectors into C57BL/6 mice and monitored the progression of the resulting BCL2-driven lymphomas. Our preliminary results indicate that high AID expression is correlated with a more aggressive phenotype of the disease. We are currently analyzing the epigenetic targets of AID in both normal GCBs and tumors, in order to find genes that could be epigenetically deregulated and contribute to the formation of lymphomas. Our results demonstrate for the first time that AID functions as a demethylase in GCBs in vivo and suggest that the epigenetic role of AID could contribute to lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

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AICDA Introduces Epigenetic Plasticity in Germinal Center-Derived Lymphomas and Accelerates Lymphomagenesis

Blood ◽

10.1182/blood.v128.22.1045.1045 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1045-1045 ◽

Cited By ~ 1

Author(s):

Maria Del Pilar Dominguez ◽

Matt Teater ◽

David Redmond ◽

Zhengming Cheng ◽

Jayanta Chaudhuri ◽

...

Keyword(s):

Dna Methylation ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Cytosine Methylation ◽

Gene Set Enrichment Analysis ◽

Dna Hypomethylation ◽

B Cell Lymphomas ◽

Lower Expression ◽

Epigenetic Plasticity

Abstract Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors derived from germinal center B cells (GC) B cells. Previous work from our group established that inferior outcome in DLBCL is associated with higher degrees of intra-tumor and inter-tumor cytosine methylation heterogeneity, although the molecules driving this epigenetic perturbation are unknown. We investigated the contribution of activation-induced cytidine deaminase (AICDA) to cytosine methylation heterogeneity in DLBCLs. AICDA is highly expressed in GC B cells where it drives somatic hypermutation (SHM) of immunoglobulin (Ig) and non-Ig genes. AICDA is also expressed in DLBCLs and high level of AICDA in CHOP-treated DLBCL patients is associated with unfavorable prognosis. In addition to mutagenesis, AICDA can cause DNA demethylation through the removal of modified cytosine residues. Indeed, we previously reported that GC B cells manifest a marked AICDA-mediated DNA hypomethylation signature as well as a high degree of epigenetic heterogeneity as compared to other B-cell subsets. Thus, we hypothesized that AICDA could contribute to the progression of GC-derived lymphomas by facilitating epigenetic plasticity through the redistribution of cytosine methylation. We overexpressed AICDA in VavP-Bcl2 transgenic mice -which develop B cell lymphomas of GC origin- using an AICDA-expressing retroviral vector (VavP-Bcl2+Aicda) vs empty vector (VavP-Bcl2) as control and transplanted them into lethally irradiated recipient mice. We observed more aggressive lymphomas based on greater disruption of the splenic architecture and higher degree of B cell infiltration in organs such as lung, liver and kidney in VavP-Bcl2+Aicda mice (n=7) compared to VavP-Bcl2 mice (n=6). Notably, the overexpression of AICDA reduced significantly the lifespan of the mice (Log-rank test p=0.0289) with a median survival of 214 days for VavP-Bcl2+Aicda mice (n=10) and 293 days for VavP-Bcl2 animals (n=9). Neoplastic B cells from VavP-Bcl2+Aicda and VavP-Bcl2 micedisplayed similar SHM rate in Ig genes (JH4 and Sm) and AICDA off-targets (Bcl6, Cd83 and Pax5), suggesting that the more aggressive phenotype is not likely due to increased mutagenesis. We then profiled the DNA methylation landscape of these lymphomas using enhanced reduced representation bisulfite sequencing. We analyzed the DNA methylation level and heterogeneity of all represented CpGs by calculating the mean methylation difference and interquartile range (IQR) between VavP-Bcl2+Aicda and VavP-Bcl2 tumors. A principal component analysis of all CpGs, each one represented by its mean methylation and IQR differences across replicates, revealed methylation loss and increased intertumor heterogeneity within VavP-Bcl2+Aicda methylomes compared to VavP-Bcl2. AICDA-perturbed CpGs (n=49,750) in particular showed significant intratumor heterogeneity (p<1e-300) in AICDA overexpressing tumors. These altered CpGs were depleted in promoters and enriched in introns and intergenic regions. We observed a remarkably similar pattern of hypomethylation and increased methylation heterogeneity in Aicda+/+ GC B cells compared to Aicda-/- GC B cells (AICDA-perturbed CpGs n=64,323) and also in primary DLBCLs with high AID compared to low AID expression (AICDA-perturbed CpGs n=37,557), suggesting a conserved epigenetic function of AICDA in GC B cells and human and mouse GC-derived lymphomas. We performed RNA sequencing on BCL2-driven lymphomas and found that expressed genes containing AICDA-perturbed CpGs displayed significantly lower expression in VavP-Bcl2+Aicda compared to VavP-Bcl2 tumors (gene set enrichment analysis (GSEA) NES=-1.68, FDR<0.001). Pathway analysis revealed that these AICDA-epigenetic target genes were associated with MAPK- and KMT2D-signaling pathways, which are related to B cell activation and lymphomagenesis. Similarly to mouse tumors, genes enriched for AICDA-perturbed CpG in human lymphomas exhibited lower expression in AID high DLBCLs compared to AICDA low DLBCLs (GSEA NES=-1.65, FDR<0.001) and were associated with MAPK and KMT2D pathways. These results demonstrate that AICDA acts as a methylome modifier in GC-derived lymphomas, introducing epigenetic plasticity and leading to gene expression changes, thus contributing to clonal evolution and higher adaptability to an evolving environment. Disclosures Melnick: Janssen: Research Funding.

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DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis.

Blood ◽

10.1182/blood.v110.11.685.685 ◽

2007 ◽

Vol 110 (11) ◽

pp. 685-685 ◽

Cited By ~ 1

Author(s):

Rita Shaknovich ◽

Leandro Cerchietti ◽

Maria E. Figueroa ◽

Ari Melnick

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Dna Methyltransferase ◽

Critical Role ◽

Epigenetic Programming ◽

Differential Digestion ◽

Epigenetic Signatures

Abstract Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs.

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Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽

10.1182/blood.v122.21.4943.4943 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4943-4943

Author(s):

Charles Repetti ◽

Hsueh-Hua Chen ◽

Yongbao Wang ◽

Vanessa A Jones ◽

Albert K Ho ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Somatic Mutation ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Sequence Variant ◽

Low Grade ◽

Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15). Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

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The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling

Blood ◽

10.1182/blood-2019-131641 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 79-79

Author(s):

Zev J. Greenberg ◽

Darlene A. Monlish ◽

Rachel L. Bartnett ◽

Jeffrey J. Bednarski ◽

Laura G. Schuettpelz

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Development ◽

B Cell Development ◽

Cellular Migration ◽

Physical Interaction ◽

Hematopoietic Stem ◽

Human Phenotype

The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

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Genome-Wide DNA Methylation Landscape Defines IGHV Mutated and Unmutated B Cell Chronic Lymphocytic Leukemias

Blood ◽

10.1182/blood.v120.21.526.526 ◽

2012 ◽

Vol 120 (21) ◽

pp. 526-526

Author(s):

Junfeng Luo ◽

Justin Choi ◽

Lirong Pei ◽

Farrukh Awan ◽

Eun-Joon Lee ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

B Cells ◽

B Cell ◽

Dna Hypomethylation ◽

Excellent Correlation ◽

Methylation Analysis ◽

Promoter Regions ◽

Genome Wide ◽

Dna Methylation Analysis

Abstract Abstract 526 Chronic lymphocytic leukemia (CLL) is a biologically and clinically heterogeneous disease. The somatic hypermutation status of the immunoglobulin heavy chain variable (IGHV) genes has been identified as one of the most robust prognostic markers in CLL. Patients with unmutated IGHV status (U-CLL) typically experience an inferior outcome compared to those whose clones express mutated IGHV genes (M-CLL). We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from a group of 43 CLL patients using reduced representation bisulfite sequencing (RRBS). Using base-pair resolution methylation sequencing, 2323 differentially methylated regions between CLL and normal B cells (CLL-specific DMRs) and 569 between M-CLL and U-CLL samples (IGHV-specific DMRs) were identified in the CLL genomes. The IGHV-specific DMRs are mostly unique when compared to the CLL-specific DMRs. Less than 10% of the IGHV-specific DMRs are located in promoter regions; however, more than half of these overlap with known DNase I hypersensitive sites, enhancer regions marked by histone modification (H3K4Me1 and H3K27Ac), and transcription factor binding sites in the ENCODE datasets, which indicates that these DMRs contain regulatory sequences. Distinctive DNA methylation patterns were observed in M-CLL and U-CLL samples. Overall, U-CLL was found to contain 50% more hypermethylated regions than M-CLL samples. The hypermethylated loci observed in the U-CLL samples also appear to be hypermethylated in normal naïve B cells as compared memory B cells, suggesting that M-CLL and U-CLL differ in differentiation status corresponding to normal B cell differentiation stages. RNA-seq analysis performed using matched samples (n=34), in which both DNA methylation and gene expression data were available, demonstrated excellent correlation between DNA methylation and gene expression. Several genes whose expression status was previously shown to be associated with CLL prognosis such as ZAP70, CRY1, LDOC1, SEPT10, LAG3, and LPL were differentially methylated in the promoter regions between M-CLL and U-CLL samples indicating that DNA methylation plays an important role in defining the gene expression patterns of these prognostic genes. We further validated 9 genes with IGHV-specific DMRs in the promoter regions using bisulfite pyrosequencing, and the results demonstrated excellent correlation between differential methylation and IGHV mutation status. These novel differentially methylated genes could be developed into biomarkers for CLL prognosis. In addition, DNA hypomethylation was observed in a significant number of genes involved in lymphocyte activation such as PDCD1, NFATc1, and CD5. DNA hypomethylation was observed in the proximal promoter and far up-stream enhancer regions of CD5, an important cell surface marker that uniquely identifies CLL. Overall, the DNA methylation landscape in CLL patients indicates that CLL B cells possess an active B-cell phenotype; at the same time, U-CLL and M-CLL are faithfully committed to their lineage resembling either naïve or memory B cells. In summary, this comprehensive DNA methylation analysis has identified a large number of novel epigenetic changes in CLL patients. The results from this study will further advance our understanding of the epigenetic contribution to molecular subtypes in CLL. Disclosures: No relevant conflicts of interest to declare.

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In Vitro Study Of The Mechanisms Involved In The Bone Marrow Mesenchymal Stromal Cell Modulatory Effect On B Cell Function

Blood ◽

10.1182/blood.v122.21.1053.1053 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1053-1053

Author(s):

Eliana Amati ◽

Giulio Bassi ◽

Mariano Di Trapani ◽

Francesco Liotta ◽

Francesco Annunziato ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

B Cells ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Conflicts Of Interest ◽

Cpg Oligodeoxynucleotide ◽

Ifn Γ ◽

Cell Inhibition

Abstract Human bone marrow Mesenchymal Stromal Cells (MSC) are potent modulators of T cell activation and proliferation, mainly through the production of partially defined soluble factors, including the IFN-g-induced tryptophan-degrading enzyme IDO, a key immunosuppressive effector pathway. Actually, MSC may affect the functions of virtually all immune effector cells, including B cells. However, current literature concerning MSC immunomodulatory activity on B cells is still controversial, due to both biological peculiarities of B cells, which do not produce IFN-γ, a key MSC-triggering cytokine, and to different and poorly comparable experimental approaches. Human purified B cells, either resting or activated for 4 days with a stimulation cocktail (CD40 ligand + enhancer polyhistidine mAb MAB050 + rhIL-2 + mouse F(ab’)2 anti-human IgM/IgA/IgG + CpG oligodeoxynucleotide 2006) were co-cultured with MSC, either at resting conditions or following inflammatory priming (MSC pre-incubation with IFN-γ + TNF-α for 48 hours), or with MSC supernatants. CD27-positive (memory) and CD27-negative (naïve) B cell survival, proliferation, and intracellular activation status (through signaling network analysis by Phosphoflow) were assessed. Our results showed that MSC are normally supportive cells, not intrinsically capable of suppressing B cell proliferation, and require inflammatory priming to acquire B cell inhibitory potential. Inflammatory-primed MSC impair significantly activated B cell growth in a cell contact-independent manner, as supernatant is sufficient to provide maximal inhibition of B cell proliferation. B cell inhibition by MSC is not related to either induction of B cell apoptosis or early signaling events necessary for B cell activation. In addition, IDO pathway triggered in IFN-γ-primed MSC seems to have a role also in B cell inhibition by interfering with the tryptophan metabolism. Overall, B cell behavior following the interaction with MSC depends on the functional state of both B cells and MSC. The role of IDO in B cell regulation needs further investigation, as it may be relevant to develop new therapeutic approaches in pathological conditions related to B cell hyper-activation. 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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Infectious Origins of Childhood Leukemia

Blood ◽

10.1182/blood.v118.21.751.751 ◽

2011 ◽

Vol 118 (21) ◽

pp. 751-751

Author(s):

Lars Klemm ◽

Srividya Swaminathan ◽

Anthony M Ford ◽

Klaus Schwarz ◽

David G. Schatz ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

Childhood All ◽

Leukemic Clone ◽

Germinal Center B Cells

Abstract Abstract 751 Background: In most cases, childhood acute lymphoblastic leukemia can be retraced to a recurrent genetic lesion in utero, which establishes a pre-leukemic clone. The TEL-AML1 fusion gene, for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only <1% of these children will eventually develop leukemia. Encounter of infectious antigen in B cell typically leads to activation of the mutator enzyme AID. While AID is required for class switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of germinal center B cells, its premature activation may be deleterious. The underlying questions for this project were (1) how are B cells during their early development safeguarded from pre-mature AID expression and (2) whether pre-mature expression of AID in early B cell development is deleterious in the sense that it pre-disposes to the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results: We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Ra suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Ra signaling involves Stat5 phosphorylation, we studied inducible Cre-mediated deletion of Stat5, which had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Ra/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Ra/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Ra surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Ra-positive Fraction C' pre-B cells to IL7Ra-negative Fraction D pre-B cells. Conclusion: AID is a tightly controlled mutator enzyme in mature germinal center B cells. The factors that prevent premature expression of AID during early B cell development were not known. Here, we here we report a novel, IL7Ra/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Ra/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL. Disclosures: No relevant conflicts of interest to declare.

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The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽

10.1182/blood.v114.22.1465.1465 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1465-1465

Author(s):

Jason Mullenix ◽

Kimi Y Kong ◽

Kristin Severns Owens ◽

Jason Rogers ◽

Shannon FitzPatrick ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Mature Mirnas ◽

Cell Development ◽

B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

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Demylinating Peripheral Neuropathy and Lymphoplasmacytic Lymphoma

Blood ◽

10.1182/blood.v116.21.5090.5090 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5090-5090

Author(s):

Carol Ann Huff ◽

Vinay Chaudhry ◽

Charlotte Sumner ◽

David Cornblath

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Peripheral Neuropathy ◽

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Demyelinating Polyneuropathy ◽

Lymphoplasmacytic Lymphoma ◽

Modest Improvement ◽

Bone Marrow Biopsies

Abstract Abstract 5090 Lymphoplasmacytic lymphoma (LPL) is a neoplasm of small B lymphocytes, plasmacytoid lymphocytes, and plasma cells, which does not fulfill criteria for any of the other small B-cell lymphoid neoplasms (WHO 2008). Neuropathy has been described in association with Waldenstrom's macroglobulinemia, but less so with LPL. We present 7 cases of neuropathy and LPL to highlight the variable presentations. 1) A 56 year old man developed sensory ataxia with an IgM kappa non-MAG paraprotein. CSF protein was 97. Stabilized on MMF, he slowly worsened. Plasma exchange (PE) was given with improvement. Bone marrow (BM) 4 years into his course revealed LPL. Rituximab was given, and his PE reduced. 2) A 49 year old woman developed progressive weakness with rapid decline in January 2010. NCS showed demyelinating polyneuropathy, and CSF protein was 179. An IgM kappa non-MAG paraprotein was found. BM was normal. PE was given with improvement but later prednisone, IVIg and rituximab twice did not help. Repeat BM revealed 2% clonal CD20+ CD5negCD10neg B cells by flow cytometry. PE was given with modest improvement. Cyclophosphamide 1 gm/m2 monthly was given with improvement. 3) A 53 year old man noted imbalance and distal weakness. NCS showed absent SAPs and prolonged distal and F wave latencies. An anti-MAG positive, IgM kappa paraprotein was found. In 2002 BM was normal. He received rituximab weekly × 4 doses. 7 years later, he developed anemia and worsening neuropathy. Repeat BM showed 0.5% CD20+CD5negCD10neg kappa-restricted B cells by flow cytometry and weekly rituximab was reinitiated. 4) A 62 year old man developed weakness and areflexia. NCS showed asymmetric demyelinating polyneuropathy. Biclonal gammopathy of IgM kappa and IgG kappa was found. BM showed LPL by histopathology. Prednisone was given with improvement. Later two courses of weekly rituximab were given. 5) A 55 year old woman developed asymmetric weakness. NCS showed asymmetric demyelinating polyneuropathy. MRI showed enlargement, abnormal signal intensity, and abnormal enhancement of bilateral radial, median, and ulnar nerves. She was found to have an IgG kappa paraprotein and LPL on BM biopsy. She was treated with rituximab, cyclophosphamide, fludarabine, and PE. In each case, the primary feature driving the need for therapy was the neuropathy and not the underlying hematologic process. Further, worsening neuropathy in 3 cases led to repeat bone marrow biopsies revealing a clonal B cell process and a diagnosis of lymphoplasmacytic lymphoma. Thus, in the presence of an IgM monoclonal gammopathy and peripheral neuropathy, we suggest bone marrow examination for LPL and then consideration of therapy directed toward the abnormal B cell clone. Disclosures: No relevant conflicts of interest to declare.

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