Iterative Germinal Center Re-Entries of Memory B-Cells with t(14;18) Translocation and Early Steps of Follicular Lymphoma Progression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 150-150
Author(s):  
Sandrine Roulland ◽  
Jocelyne Stephanie Sungalee ◽  
Ester Morgado ◽  
Emilie Mamessier ◽  
Emilie Gregoire ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Lymph Nodes ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Systemic Disease ◽  
Memory B Cells ◽  
Lymphoid Organs ◽  
Healthy Humans

Abstract Abstract 150 The recent demonstration that memory B-cells can re-enter germinal centers (GCs) and participate to new rounds of GC reactions has opened the possibility that multi-hit B-cell lymphomagenesis could be a much more dynamic process than initially anticipated, gradually progressing throughout the successive passages of memory B-cells in GCs during a lifetime of successive immunological challenges. Here, we provide evidence for this scenario in follicular lymphoma (FL), a GC derived B-cell malignancy initiated in the bone marrow by the hallmark t(14;18) BCL2/IGH translocation. To address this issue, we engineered an original sporadic BCL2tracer mouse model mimicking the rare occurrence of t(14;18) translocation in humans through V(D)J recombination errors (1 in a million B-cells) allowing to track the resultant BCL2-expressing clones; and underwent a molecular/immunofluorescent tracking of t(14;18)+ clones vs. normal memory B-cells in paired lymphoid tissue samples (spleen, lymph nodes, bone marrow) from healthy individuals. We first show that contrary to the current dogma, ectopic BCL2 expression is not sufficient to provoke the FL's characteristic differentiation arrest of activated B-cells as GC B-cells, thereby suggesting that differentiated BCL2+ memory B-cells must return to the GC to acquire additional oncogenic hits and “fix” in situ growth. Strikingly, we further find that in a small fraction of “healthy” humans, such differentiation arrest already operated, and that a clonally expanded population of t(14;18)+ cells with FL-like features have widely disseminated in blood and in multiple lymphoid organs (spleen, lymph nodes, bone marrow), with unprecedentedly reported frequencies (from 1/million to 1/500 cells in some individuals), shaping the systemic disease presentation observed in FL patients. Using molecular/immunofluorescent backtracking of such clones in various paired and remote lymphoid organs, we further demonstrate that t(14;18)+ clones systematically display an extensive history of AID (activation-induced cytidine deaminase)-mediated events compatible with iterative rounds of GC co-opting, in sharp contrast to single memory B-cell clones from the same individuals. We thus show that BCL2-expressing memory B-cells require multiple GC transits to acquire the distinctive FL-like maturation arrest as centrocyte/centroblasts and to progress to advanced FL precursor stages. Altogether, our data argue for a model of lymphomagenesis, in which progression from precursor stages to FL occurs asymptomatically over an extended period of time by subverting the dynamic and plastic attributes of memory B-cells. This understanding of the pre-clinical phases driving FL development in asymptomatic patients should help rationalize prospective approaches designed to identify biomarkers of risk, and innovative therapeutic targets present in early, potentially more curable phases of the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Follicular Lymphoma Cell Niche: Identification of a Preeminent IL-4-Dependent TFH- B Cell Axis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 757-757
Author(s):  
Karin Tarte ◽  
Pangault Celine ◽  
Patricia Ame-Thomas ◽  
Philippe Ruminy ◽  
Delphine Rossille ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Lymph Nodes ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Immune Response Gene ◽  
Cell Compartment ◽  
Cell Axis

Abstract Abstract 757 Follicular lymphoma (FL) B cells contract tight connections with their microenvironment, which governs the pathogenesis and progression of the disease. Indeed, specific immune response gene signatures, obtained on whole biopsy samples, have been associated with patient survival, independently of classical clinical features. In this study we performed, using Affymetrix U133 Plus 2.0 oligonucleotide microarrays, the gene expression profiling of purified CD19pos B-cell and CD19negCD22neg non-B cell compartments, prospectively obtained from FL and reactive lymph nodes. Unsupervised analyses of B-cell compartment in one hand (n=21) and non-B cell compartment in the other hand (n=14) allowed to differentiate FL from reactive samples. We then identified 677 nonredundant genes defining the FL synapse, i.e. the list of genes involved in the crosstalk between B cells and their microenvironment in FL. Using Ingenuity pathway analysis we pointed out 26 FL-specific functional networks among this FL synapse, including an IL-4-centered pathway. Interestingly, whereas in tonsils and reactive lymph nodes only exceptional scattered phospho-STAT6pos cells were observed, a high number of CD20posphospho-STAT6pos cells were evidenced within FL biopsies. In addition, several IL-4-target genes, including IL4I1 and HOXC4, were overexpressed in malignant B cells. Altogether, these results demonstrated that the upregulation of IL-4 within FL microenvironment was associated to a strong activation of FL B cells. In addition, FL microenvironment was characterized by a strong enrichment in follicular helper T cells (TFH), as demonstrated through both Gene Set Enrichment Analysis (GSEA)-based transcriptomic approach and flow cytometry analysis of the CD4posCXCR5hiICOShi cell compartment. The majority of phospho-STAT6pos B cells were located at the vicinity of cells expressing the PD-1 TFH marker. Moreover, purified FL-derived TFH, unlike non-TFH T cells, expressed IL4. Finally, whereas chronically inflamed tonsils also exhibited an increase percentage of TFH cells, tonsil-derived TFH did not express IL4. Altogether, our study demonstrated that tumor-infiltrating TFH specifically express functional IL-4 in FL, creating therefore an IL-4-dependent TFH-B cell axis. This crosstalk could sustain FL pathogenesis and represent a new potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Follicular Lymphoma-Like B Cells In Healthy Individuals Are Released From Pretumoral Niches Established In Secondary Lymphoid Tissues

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 466-466 ◽  
Author(s):  
Sandrine Roulland ◽  
Stephanie Sungalee ◽  
Emilie Gregoire ◽  
Fabien Guilloton ◽  
Ester Morgado ◽  
...  
Keyword(s):  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Genomic Instability ◽  
Malignant Transformation ◽  
Conflicts Of Interest ◽  
Lymphoid Organs ◽  
Early Event ◽  
Lymphoid Tissues ◽  
Healthy Individuals

Abstract Abstract 466 Follicular Lymphoma (FL) is a frequent mature B-cell neoplasia resulting from the malignant transformation of germinal center (GC) B-cells in secondary lymphoid organs. The t(14;18) translocation constitute both a genetic hallmark and a critical early event in the natural history of FL. t(14;18) is however not sufficient for malignant transformation, and further synergistic oncogenic events are clearly required. In line with this, t(14;18)+ cells can be detected in the blood of most healthy individuals (HI). However, large differences in frequencies can be observed between individuals with yet uncertain significance. We recently demonstrated that in HI carrying high t(14;18) frequencies, such circulating translocated cells constitute an expanding population of atypical B-cells displaying the geno/phenotypic features of FL. In particular, we found that in such individuals, these cells are “frozen” at a GC B-cell stage of differentiation, where sustained AID expression leads to constitutive somatic hypermutation (SHM), class switch recombination (CSR) and genomic instability. As such processes are responsible for the progressive acquisition of secondary oncogenic alteration in B-cell lymphomagenesis, we proposed that t(14;18)+ cells could represent bona fide FL precursors released from established premalignant niches in lymphoid organs (Roulland et al. JEM 2006, Agopian et al. 2009). To address this possibility, we now performed a systemic characterization of FL precursors in normal human biopsies: 20 paired blood/spleen/lymph nodes (LN) issued from organ donors, 10 blood/BM samples from patients undergoing cardiac surgery and 7 hyperplastic tonsils. Using a sensitive fluctuation PCR assay, we found that while children tonsils were negative, t(14;18)+ cells were very frequent in adult tissues (75% of spleens, 59% of LN and 30% of BM samples). The frequencies were distributed over a wide frequency spectrum (>500-fold), among which t(14;18)+ frequencies in spleen (up to 1/1000 cells) were surprisingly high, far above levels generally found in blood (from 1/100,000 to 1/10 million). To test the possibility that lymphoid organs (and in particular the spleen) constitute sanctuaries of FL precursors, we characterized t(14;18) cells in tissues at the molecular level, and evaluated if paired circulating and resident t(14;18)+ cells are clonally related. Using detailed molecular analysis of BCL2/IGH amplicons obtained by LR-PCR on coupled tissue samples or isolated splenic B-cell subsets discriminating naïve and GC/post-GC B-cells, we demonstrate that t(14;18) clones issued from spleen, LN and BM are the resident counterparts of the circulating pre-FL cells (including a «frozen» GC phenotype maintaining AID activity, with the presence of highly mutated Sμ regions, intra-Sμ deletions and for the most advanced clones, unusual stigmata of AID-mediated genomic instability linked to malignant progression). Furthermore, intraclonal variation (ICV) analysis of the upstream Sμ flanking region revealed the presence of an intense trafficking of the t(14;18)+ FL-like clones between lymphoid organs and/or blood. Indeed, clones presenting identical BCL2/IGH signature could be found in different organs and blood, and yet displayed systematic ICV, indicative of ongoing SHM and further supporting the derivation of such cells from GC derived B-cells with sustained AID activity. Collectively, these data establish a direct clonal relationship between resident t(14;18)+ cells and their circulating counterparts, and the early potential of such pre-FL GC-B cells to invade other reactive GC, and to disseminate in BM. These findings strongly impact on the current understanding of disease progression, and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mesenchymal Stromal Cells Orchestrate Follicular Lymphoma Cell Niche Through the CCL2-Dependent Recruitment and Polarization of Monocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1566-1566
Author(s):  
Fabien Guilloton ◽  
Gersende Caron ◽  
Cédric Ménard ◽  
Céline Pangault ◽  
Patricia Amé-Thomas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Growth ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Gene Set Enrichment Analysis ◽  
Cell Niche

Abstract Abstract 1566 Accumulating evidence indicates that infiltrating stromal cells contribute directly and indirectly to tumor growth in a wide range of solid cancers and hematological malignancies. In follicular lymphoma (FL), malignant B cells are found admixed with heterogeneous lymphoid-like stromal cells within invaded lymph nodes and bone marrow (BM). In addition, in vitro functional studies have underlined that mesenchymal cells recruit malignant FL B cells and protect them from spontaneous and drug-induced apoptosis. In particular, we have previously demonstrated that mesenchymal stromal cells (MSC) efficiently support in vitro FL B-cell survival, especially after their engagement towards lymphoid differentiation through treatment with TNF-α and Lymphotoxin-α1β2 (TNF/LT) or after coculture with malignant B cells. However, the mechanisms of this supportive activity remain largely unknown. In this study, we used Affymetrix U133 Plus 2.0 microarrays, to compare the gene expression profile (GEP) of bone marrow-derived MSC (BM-MSC) obtained from 10 FL patients at diagnosis versus 6 age-matched healthy donors (HD). In these conditions, neither the CFU-F concentration in the BM nor the cumulative population doubling of BM-MSC significantly differed between HD and FL patients. Unsupervised analysis was able to perfectly segregate FL-MSC from HD-MSC and we identified, using supervised analyzes, a list of 408 probesets defining FL-MSC signature, including 320 nonredundant genes upregulated in FL-MSC compared to HD-MSC. We then defined the GEP of human lymphoid-like stroma using HD-MSC treated in vitro by TNF/LT and demonstrated, by a Gene Set Enrichment Analysis (GSEA) approach, that the FL-MSC signature is significantly enriched for genes associated with a lymphoid-like commitment. Interestingly, CCL2 was strongly overexpressed by FL-MSC, was upregulated in HD-MSC by coculture with malignant B cells, and was detected at a higher level in FL BM plasma compared to normal BM plasma (504.4 pg/mL [23.8-4413] versus 33.9 pg/mL [5-126.1]; P <.01). In agreement, FL-MSC triggered a more potent CCL2-dependent monocyte migration than HD-MSC. Moreover, FL-MSC and macrophages cooperated to sustain malignant B-cell growth through both protection from apoptosis and enhancement of cell proliferation. Finally, FL-MSC promoted monocyte differentiation towards a proangiogenic LPS-unresponsive phenotype close to that of tumor-associated macrophages. We unraveled a key role for the Notch pathway in this process and identified an overexpression of JAGGED1 in FL-MSC compared to HD-MSC. Altogether, these results highlight the complex role of FL stromal cells that promote direct tumor B-cell growth and orchestrate FL cell niche. The identification and characterization of this intricate network of cell interactions may provide novel therapeutic targets in this disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Antigen-Specific B Cell Memory

2000 ◽  
Vol 191 (7) ◽  
pp. 1149-1166 ◽  
Author(s):  
Louise J. McHeyzer-Williams ◽  
Melinda Cool ◽  
Michael G. McHeyzer-Williams
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
Cell Memory ◽  
Specific Memory ◽  
B Cell Memory ◽  
Cellular Components

The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220−CD138−) that are distinct from antibody-secreting B cells (B220+/−CD138+) and B220+CD138− memory B cells. These nonsecreting somatically mutated B220− memory responders rapidly dominate the splenic response and comprise &gt;95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75–85%) and the bone marrow (&gt;95%) expresses the B220− phenotype. Upon adoptive transfer, B220− memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220+ counterparts. The pattern of cellular differentiation after transfer indicates that B220− memory B cells act as stable self-replenishing intermediates that arise from B220+ memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220− compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long-term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function.

CD40 Ligand Activation Alters B Cell Migration to Secondary Lymphoid Organs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 935-935
Author(s):  
Yvonne A. Efebera ◽  
Tahamtan Ahmadi ◽  
Amanda Flies ◽  
David H. Sherr
Keyword(s):  
B Cells ◽  
Lymph Nodes ◽  
B Cell ◽  
Cd40 Ligand ◽  
Lymphoid Organs ◽  
Cell Populations ◽  
Secondary Lymphoid Organs ◽  
Activated B Cells

Abstract Background: An increased understanding of the requirements for antigen presentation has encouraged development of cell-based cancer vaccines. Trials using dendritic cells (DC) as antigen presenting cells (APC) for immunotherapy of several malignancies have shown considerable success. However, the difficulty in generating large numbers of DC required for these immunizations has led to the search for alternative APC. One such candidate is the CD40 ligand (CD40L)-activated B cell, populations of which can readily be expanded in vitro. To be an effective vehicle for antigen presentation to T cells, CD40L-activated B cells must be capable of migrating to secondary lymphoid organs. Therefore, CD40L-activated B cell migration following subcutaneous or intravenous injection was evaluated. Methods: Splenic B cells from GFP transgenic mice were activated with CD40L + IL-4 and expanded in vitro prior to i.v. or s.c. injection of 3–4 x 107 into C57BL/6 mice. Recipient mice were sacrificed 2 hrs or 1–14 days thereafter and the percentage of GFP+/B220+ B cells quantified in spleens and lymph nodes by flow cytometry. Localization of these cells within lymphoid organs was determined by immunohistochemistry. In some experiments, activated C57BL/6 B cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) to evaluate cell growth in vivo. Results: Murine B cell populations were readily expanded by culture on CD40L-transfected L cells in the presence of IL-4. CD40L-activated B cells expressed high levels of CD80, CD86, and LFA-1 but decreased levels of L-selectin relative to naive cells. Following i.v. injection, activated B cells were detected in spleens and lymph nodes within 1 day. Peak concentrations of activated B cells were noted in spleens and lymph nodes on days 7 (4.8% of injected cells) and 10 (1.25% of injected cells) respectively, suggesting expansion of the activated B cell population in vivo. Naive B cells injected i.v. were detected within 1 day but their number declined precipitously thereafter. Following s.c. injection, peak levels of CD40L-activated B cells were noted on day 5 (spleens) and day 7 (lymph nodes). As determined by immunohistochemistry, both CD40L-activated and naïve B cells injected i.v. appeared in B cell regions of spleens and lymph nodes. While the kinetics of accumulation of CD40L-activated B cells injected s.c. or i.v. were similar, s.c. injected CD40L-activated B cells homed to the T cell regions of spleens and lymph nodes. CFSE experiments indicated that these activated B cells continue to grow in vivo. In contrast, naïve B cells injected s.c. only appeared in B cell regions. Conclusion: CD40L-activated B cell populations can readily be expanded in vitro, CD40L-activated B cells migrate to secondary lymphoid organs even when injected s.c., activated B cell populations expand in vivo, and s.c. injected, CD40L-activated B cells preferentially home to T cell regions of secondary lymphoid organs. These results suggest that this effective APC may serve as an important vehicle for delivery and presentation of exogenous (e.g. tumor) antigens to T cells in vivo.

ZAP-70 Enhances Migration of Malignant B Cells towards Lymphoid Organs in a Burkitt Lymphoma Xenograft Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2844-2844
Author(s):  
Noelia Purroy ◽  
Eva Calpe ◽  
Pau Abrisqueta ◽  
Cecilia Carpio ◽  
Carles Palacio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Xenograft Model ◽  
Lymphoid Organs ◽  
Transfected Cells

Abstract Abstract 2844 Introduction. ZAP-70 (ξ-associated protein) is a protein tyrosine kinase of the Syk/ZAP family that plays a crucial role in cellular activation in T and NK cells. High expression of ZAP-70 protein in malignant cells from Chronic Lymphocytic Leukemia (CLL) correlates with adverse clinical prognostic features, such as unmutated IgHV genes, short time to progression, and short survival. Moreover, ZAP-70 protein has been related to aggressive features of the CLL cells, such as enhanced B-cell receptor (BCR) signaling and higher migration capacity. To further investigate into the mechanisms by which ZAP-70 protein influences the clinical outcome of patients with CLL, we analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells. For this, Ramos and Raji (Burkitt) B-cell lines were stably transfected with a ZAP-70 expressing vector (pEGFP-N2ZAP-70). Raji transfectant showed constitutively phosphorylated ZAP-70 protein, whilst Ramos cells required stimulation with 5 μg/ml F(ab') 2 anti-IgM to get ZAP-70 activated. ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21 (own data, Blood 2011 pre-published). CCR7 ligands (chemokines CCL21 and CCL19) are mainly expressed in high endothelial venules and the T zones from secondary lymphoid organs. The aims of this study were firstly to evaluate in vivo the migratory/invasive capability of pEGFP-N2ZAP-70 transfected Raji and Ramos cell lines compared to pEGFP Raji and Ramos cell lines; and later, to compare the overall survival (OS) of mice injected with pEGFP-N2ZAP-70 transfected cells to those injected with only pEGFP transfected cells. Methods. For this, a total of 27 7- to 8-week old SCID (CB17Crl) mice were used. Mice were inoculated intravenously with 5×106 cells of each cell line (6 mice with Raji-GFP, 5 mice with Raji-GFP-ZAP-70, 5 mice with Ramos-GFP and 10 mice with Ramos-GFP-ZAP-70). Mice were observed for the onset of hind legs paralysis, dyspnea, or evidence of tumor growth, once symptoms appeared, mice were euthanized and lymphoid and non-lymphoid organs were obtained for further analysis of the presence of GFP-positive cells by flow cytometry and immunohistochemistry. Results. Twenty-six out of twenty-seven injected mice were included in the analysis. The excluded mouse was found dead before it could be euthanized to obtain the organs. In the Raji xenograft model, 11/11 (100%) of mice had hind legs paralysis as the first symptom to appear. The median survival was 19 days for GFP-ZAP-70 and 16 days for GFP injected mice. There were no statistically significant differences between survival of GFP-ZAP-70 and GFP injected mice (OS was 66.7% [95% CI 38.4–100] vs 33.3% [95% CI 0–71.1], p=0.784, at 19 and 16 days, respectively). In the Ramos xenograft model, 6/15 (40%) of mice showed hind legs paralysis as the first symptom to appear, as well as evidence of abdominal tumor growth in 6/15 (40%), whereas in 3/15 (20%) the established event was dyspnea. The median survival in Ramos xenograft model was 40 days for GFP-ZAP-70 and 38 days for GFP injected mice. Again there were no statistically significant differences between survival of GFP-ZAP-70 and GFP Ramos injected mice (OS was 50% [95% CI 18.4–81.6] vs 40% [95% CI 0–83.8], p=0.180, at 40 and 38 days, respectively). By flow cytometry analysis of GFP cells we found that in the Raji xenograft model there were statistically significant differences between the migration of GFP-ZAP-70 and GFP injected cells towards bone marrow (21.5% vs 5.17, p=0.011), spleen (0.08% vs 0.01%, p=0.006) and thymus (0.00% vs 0.02%, p=0.037). The highest percentages of GFP positive cells were found in bone marrow samples (mean, 9.85%), whereas in spleen and thymus the percentages of GFP positive cells were all below 0, 1%. There was no statistically significant difference between the cellular migration in the Ramos xenograft model in any of the organs analyzed. Conclusion. In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein show enhanced ability to migrate towards and infiltrate lymphoid organs in a xenograft model, specially the bone marrow, although it does not translate into a worse survival of the animals. Further specific immunohistochemical assays to determine infiltrated areas by ZAP-70 expressing lymphocytes are in process. Disclosures: No relevant conflicts of interest to declare.

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 ◽  
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.

scholarly journals Analysis of the heterogeneity of the BCR H-CDR3 repertoire in the bone marrow and spleen of 3-, 12-, and 20-month old mice 

2020 ◽  
Author(s):  
Lina Ma ◽  
Xinsheng Yao ◽  
Tao Xinxin ◽  
He Xiaoyan ◽  
Wang Peng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
Mouse Bone Marrow ◽  
Aged Mice ◽  
Cell Immunity ◽  
Repertoire Diversity ◽  
Old Mice ◽  
Peripheral B Cells

Abstract The number of central and peripheral B cells and their responsiveness are decreased in aged mice. The diversity of mouse central and peripheral B cell repertoires with increasing age has not been elucidated. In this study, we demonstrated that there were significant differences in the usage of some V, D, and J genes in the BCR H-CDR3 repertoire of bone marrow B cells, spleen B cells and spleen memory B cells in 3-, 12-, and 20-month-old mice. In the productive, pseudogene, and out-of-frame sequences, bone marrow B cells had significant differences in 5′J trimming with age; peripheral spleen B cells and memory B cells had significant differences in N1 insertion, N2 insertion, P5'D insertion, and 5'D trimming with age. The BCR H-CDR3 repertoire diversity of mouse bone marrow B cells, spleen B cells and spleen memory B cells decreased with increasing age. The proportion of overlap in bone marrow and spleen B cells, but not spleen memory B cells, of mice at different ages was lower at 3 months than at 12 and 20 months. This study is the first to report the homogeneity and heterogeneity of the CDR3 repertoire of central and peripheral B cells change as mice age, to further investigation of the decline and response of B cell immunity in young/middle/old-aged mice.

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

Blood ◽  
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&lt;0.005), splenic (35% fewer, p&lt;0.05), lymphatic (65% fewer, p&lt;0.0001), and peripheral (30% fewer, p&lt;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&lt;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&lt;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.

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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 59-59 ◽  
Author(s):  
Maria Del Pilar Dominguez ◽  
Matt Teater ◽  
Nyasha Chambwe ◽  
David Redmond ◽  
Bao Vuong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cytosine Methylation ◽  
Conflicts Of Interest ◽  
Retroviral Vectors ◽  
Dna Hypomethylation ◽  
Demethylase Activity

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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