scholarly journals Rearrangement of exogenous immunoglobulin VH and DJH gene segments after retroviral transduction into immature lymphoid cell lines.

1988 ◽  
Vol 167 (2) ◽  
pp. 372-388 ◽  
Author(s):  
S V Desiderio ◽  
K R Wolff
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Common Mechanism ◽  
Chain Gene ◽  
B Cell Differentiation ◽  
Retroviral Transduction ◽  
Flanking Sequences ◽  
B Cell Precursors ◽  
Nucleotide Sequence Determination

A model substrate for the joining of Ig VH and DJH elements has been constructed in a retroviral vector carrying a selectable marker whose expression is independent of the arrangement of the resident Ig gene segments. The substrate was introduced into lymphoid and nonlymphoid cells, and site-specific recombination between the VH and DJH elements was monitored by a direct hybridization assay. Joining of the exogenous gene segments was observed in cell lines representative of three distinct stages in early B cell differentiation. Rearrangement was not observed in three cell lines derived from mature B cells, or in a fibroblastoid cell line. The VH and DJH elements were initially arranged so that the VH-DJH junction and the recombined flanking sequences could be recovered after rearrangement. By molecular cloning and nucleotide sequence determination, VH-DJH junctions formed upon rearrangement of the substrate were found to resemble closely similar junctions in functional H chain genes. The joining of VH and DJH elements was observed to be asymmetric; loss of nucleotides occurred at the coding joints, but not at the junctions between flanking sequences. Our results suggest that Ig H and L chain gene segments are joined by a common mechanism that is more active in B cell precursors than in mature B cells. These observations provide further evidence that the rearrangement of Ig gene segments occurs by a nonreciprocal recombinational mechanism. The model substrate described here is likely to be of use in defining the nucleotide sequences that mediate rearrangement and in examining the developmental specificity of this process.

scholarly journals Ras Mediates Effector Pathways Responsible for Pre-B Cell Survival, Which Is Essential for the Developmental Progression to the Late Pre-B Cell Stage

2000 ◽  
Vol 192 (2) ◽  
pp. 171-182 ◽  
Author(s):  
Hitoshi Nagaoka ◽  
Yoshimasa Takahashi ◽  
Reiko Hayashi ◽  
Tohru Nakamura ◽  
Kumiko Ishii ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Chain Gene ◽  
B Cell Differentiation ◽  
Antigen Receptors ◽  
Cell Stage ◽  
Light Chain Gene ◽  
B Cell Survival ◽  
Effector Pathways

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21ras in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21Asn-17 Ha-ras was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21ras activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21ras activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21ras mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

scholarly journals gamma-c gene transfer into SCID X1 patients' B-cell lines restores normal high-affinity interleukin-2 receptor expression and function

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3108-3116 ◽  
Author(s):  
H Hacein-Bey ◽  
M Cavazzana-Calvo ◽  
F Le Deist ◽  
A Dautry-Varsat ◽  
C Hivroz ◽  
...  
Keyword(s):  
B Cells ◽  
Gene Transfer ◽  
B Cell ◽  
Cell Lines ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Receptor Expression ◽  
Chain Gene ◽  
High Affinity ◽  
And Function

SCID X1 is characterized by faulty T-cell and natural killer cell differentiation caused by mutation of the gamma-c chain gene encoding a number of multiple cytokine receptors (interleukin-2 [IL-2], IL-4, IL- 7, IL-9, and IL-15 receptors). To assess the feasibility of inducing long-term expression and function of the gamma-c chain, Epstein-Barr virus (EBV)-transformed B-cell lines from two patients with SCID X1 were transduced with a Moloney-derived retroviral vector containing the gamma-c chain cDNA. The viral LTR was used as the promoter. Immediately after two cycles of coculture with the psi-crip clone producing the MFG(B2)-gamma-c cDNA vector, gamma-c expression, assessed by detection of the mRNA and membrane protein expression, was found in 15% to 20% of cells. The degree of membrane expression was similar to that in control EBV-B cells. Expression increased steadily over 6 months, becoming detectable in 100% of cells, and remained stable thereafter for a total of 9 months, reflecting positive selection of transduced cells. A study of provirus integration sites showed multiple integration. The expressed gamma-c was functional, because it restored high-affinity IL- 2 receptor binding, IL-2 endocytosis, and IL-2-triggered phosphorylation of JAK-3 tyrosine kinase. Similar results were obtained with the two B-cell lines. These results show that efficient gamma-c gene transfer into B-cells lacking functional gamma-c is feasible and results in strong and stable expression of a functional gamma-c chain, apparently conferring a selective growth advantage in culture. Further in vitro studies of gamma-c gene transfer into gamma-c- hematopoietic progenitors are being conducted to assess the feasibility of correcting lymphocyte differentiation defects.

scholarly journals Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation.

1990 ◽  
Vol 10 (1) ◽  
pp. 422-425 ◽  
Author(s):  
M Briskin ◽  
M Damore ◽  
R Law ◽  
G Lee ◽  
P W Kincade ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Chain Gene ◽  
Nf Kappa B ◽  
Light Chain Gene ◽  
Immunoglobulin Kappa ◽  
Variant Cell ◽  
Late Step ◽  
Mutant Lines

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.

Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation

1990 ◽  
Vol 10 (1) ◽  
pp. 422-425
Author(s):  
M Briskin ◽  
M Damore ◽  
R Law ◽  
G Lee ◽  
P W Kincade ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Chain Gene ◽  
Nf Kappa B ◽  
Light Chain Gene ◽  
Immunoglobulin Kappa ◽  
Variant Cell ◽  
Late Step ◽  
Mutant Lines

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.

Frequent Loss of the SLP-65 Adapter Protein in Childhood Acute B-Precursor Lymphoblastic Leukemia (ALL) with TEL/AML1 Rearrangement.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 741-741 ◽  
Author(s):  
Arndt Borkhardt ◽  
Christine Damm-Welk ◽  
Thomas Wossning ◽  
Bettina Storch ◽  
Uta Fuchs ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Tumor Suppressor ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Sh2 Domain ◽  
B Cell Differentiation ◽  
Cell Leukemia ◽  
B Cell Leukemia

Abstract The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation. Disconnection between slp-65 transcripts and protein expression total slp-65 protein+ (51 patients) slp-65 protein weak (19 patients) slp-65 protein- (49 patients) PCR+ 108 51(9 TEL/AML+, 42 TEL/AML-) 19 (9 TEL/AML+, 10 TEL/AML-) 38 (15 TEL/AML+, 23 TEL/AML-) PCR- 11 0 0 11 (T-ALL)

Inhibiton of mTOR by RAD001 Restores Normal B Cell Differentiation and Prevents B Cell Lymphomas in Eμ-MYC Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2498-2498 ◽  
Author(s):  
Meaghan Wall ◽  
Gretchen Poortinga ◽  
Daniela Cardozo ◽  
Ricky W. Johnstone ◽  
Grant A. McArthur
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
B Cell ◽  
Ribosome Biogenesis ◽  
Constitutive Expression ◽  
B Cell Lymphoma ◽  
B Cell Differentiation ◽  
Days Of Therapy ◽  
B Cell Subsets ◽  
B Cell Precursors

Abstract The c-Myc proto-oncogene encodes a bHLH-LZ transcription factor that regulates proliferation, differentiation and apoptosis. Deregulated expression of c-MYC is a frequent finding in a wide variety of human cancers, including B cell lymphoma. One emerging function of c-MYC is the regulation of ribosome biogenesis, protein synthesis and metabolism i.e. cell growth. mTOR, a key downstream signal transduction molecule in the PI3K/AKT growth regulatory pathway, is amenable to pharmacological inhibition by rapamycin analogues such as RAD001. We hypothesized that control of cell growth by c-MYC is important for its ability to regulate differentiation and act as an oncogene and that RAD001, by limiting cell growth, would attenuate the transforming properties of c-MYC. In Eμ-myc mice the c-myc transgene is under the control of the immunoglobulin heavy chain enhancer (Eμ). Constitutive expression of c-MYC results in a polyclonal expansion of B cell precursors followed by lymphoma development. In the current study ‘pre-lymphomatous’ Eμ-myc mice were randomized to receive RAD001 5mg/kg (n=20) or placebo (n=18) 6 days per week from 4 weeks of age. Peripheral blood B cells were analyzed by surface marker expression after 2, 4 and 8 weeks of therapy. Mice were monitored weekly for the development of lymphadenopathy. 2 weeks of treatment with RAD001 significantly reduced the numbers of B cells in the blood of Eμ-myc mice compared to placebo (1.37±0.13 ×103/μL in the RAD001 arm versus 3.41±0.64 ×103/μL in the placebo arm, p<0.05). In particular, there was preferential suppression of less mature circulating B cell precursors over mature B cells by RAD001 resulting in B cell developmental subset profiles more closely approaching those of wild-type mice (Figure 1). Treatment with RAD001 was associated with improved lymphoma-free survival; 13/14 lymphoma-free mice (92.9%) versus 6/11 (54.6%) in the placebo group in an interim analysis of mice that had received at least 60 days of therapy. These results indicate that RAD001 can firstly oppose the expansion of B cell precursors and secondly reduce the incidence of malignant transformation induced by deregulated expression of c-MYC in B lymphocytes. These findings have implications for the application of mTOR inhibitors in the treatment or prevention of malignancies associated with MYC. Figure 1. B cell subsets after weeks of therapy Figure 1. B cell subsets after weeks of therapy

scholarly journals Interleukin-1 inhibition of B lymphopoiesis is reversible

Blood ◽  
1988 ◽  
Vol 72 (6) ◽  
pp. 2053-2055 ◽  
Author(s):  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Interleukin 1 ◽  
B Cell Differentiation ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
B Cell Precursors ◽  
Marrow Cultures

Abstract Interleukin-1 (IL-1) has multiple effects on the hematopoietic system. The present data demonstrate that IL-1 and/or products induced by it reversibly suppress B-cell differentiation. Upon the addition of 50 U/mL (2.4 ng/mL) of recombinant IL-1 alpha (rIL-1 alpha) to lymphoid long-term bone marrow cultures at their initiation, very few B lymphocytes could be detected, and the majority of cells present were myeloid. This inhibition of B lymphopoiesis did not appear to be due to effects on proliferation of mature B cells because IL-1 did not affect the proliferative response of B cells to form B-cell colonies (CFU-B). The actions of the monokine were further examined by using myeloid and lymphoid long-term bone marrow culture systems. The transfer of myeloid long-term bone marrow cultures to lymphoid conditions usually results in the cessation of myelopoiesis and initiation of B lymphopoiesis. Exposure of early B-cell precursors present under the myeloid conditions to 50 U/mL of RIL-1 did not affect their subsequent differentiation into B cells upon transfer of the cultures to lymphoid conditions. However, myelopoiesis was sustained, and B lymphopoiesis did not initiate if 50 U/mL of rIL-1 was added to myeloid bone marrow cultures at the time of their transfer to the lymphoid conditions and during biweekly feedings thereafter. Upon removal of IL-1, myelopoiesis ceased, and B lymphopoiesis initiated. Thus, the effects of IL-1 on inhibition of B lymphopoiesis are reversible.

Differentiation-Stage-Specific Expression of MicroRNAs in B-Lymphocytes and Diffuse Large B-Cell Lymphomas (DLBCL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 805-805 ◽  
Author(s):  
Raquel Malumbres ◽  
Robert Tibshirani ◽  
Elena Cubedo ◽  
Kristopher A Sarosiek ◽  
Xiaoyu Jiang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Memory B Cells ◽  
Microrna Expression ◽  
B Cell Differentiation ◽  
Cell Of Origin ◽  
Specific Expression ◽  
Differentiation Stage

Abstract B-cell development and differentiation are complex processes controlled by distinct programs of transcriptional control. A large set of transcriptional factors together or in succession control this process and their deregulation may result in block of differentiation or malignant transformation. MicroRNAs are small RNAs that orchestrate cellular functions by modulating the level of their targeted proteins by either translational arrest or transcript degradation, and play a key role in cell differentiation, apoptosis, proliferation and cancer development. An increasing number of transcription factors are being found targeted by microRNAs. Emerging evidence suggests that differentiation stage-specific expression of microRNAs occurs in the hematopoietic system and during T cell differentiation. Only limited information exists on microRNA expression in normal B cell differentiation and its malignant counterparts. Herein we analyzed microRNA expression profiles in distinct peripheral B cell differentiation stages-naïve, germinal center (GC) centroblasts and memory cells as well as tonsilar T cells. Furthermore, microRNA profiling was performed in germinal center-like (GCB-like) and activated B-cell-like (ABC-like) DLBCL cell lines originating from distinct B-cell differentiation stages. RNA, extracted with mirVana kit (AMBION) from B cell subsets and T cells enriched from normal tonsils was hybridized on LC Sciences (Houston, TX) microarrays harboring 470 human microRNAs probes (Sanger miRBase Release 9.1). Expression of selected microRNAs was confirmed by ABI RT-PCR methodology. Unsupervised clustering of microRNAs with values present in at least 50% of the samples (122 probes) resulted in perfect differentiation-stage clustering of samples. Application of Statistical Analysis of Microarrays (SAM) and Prediction Analysis of Microarrays (PAM) methods (FDR= 10%) identified a 47 microRNA cell of origin classifier for B-cells differentiation stage; 27 of these microRNAs were upregulated and 20 downregulated in centroblasts compared to memory B-cells. MicroRNAs belonging to paralog microRNA clusters (e.g. miR17-92-1, miR363-106a and miR25-106b) demonstrated similar patterns of expression in specific differentiation stages. To identify specific microRNA targets, miRanda, TargetScan and PicTar programs were used. To experimentally confirm the targets, we assessed the effects of specific microRNAs on the expression levels of targeted proteins and on the luciferase reporter under the control of the wild type and mutated 3′ UTR regions of putative target genes. Using this experimental approach we identified lymphocyte-stage-specific microRNAs which expression inversely correlated and might regulate the expression of LMO2, BLIMP1 and IRF4 proteins distinctively expressed at different differentiation stages of B lymphocytes. For example, miR223, which expression is low in GC cells but is high in naïve and memory B cells, downregulates the expression of LMO2. We next analyzed microRNA expression in DLBCL cell lines. Clustering analysis, using the 47 microRNA cell of origin classifier perfectly classified GCB-like and ABC-like cell lines. Interestingly, the expression of microRNAs in both GCB-like and ABC-like DLBCL cell lines was more similar to normal centroblasts than to memory B cells, suggesting that both may originate from distinct subpopulations of GC lymphocytes. The similarity of microRNA expression in cell lines to centroblasts was striking, with only 16 microRNAs (1 upregulated and 15 downregulated in cell lines) showing noticeable differences in levels of expression compared to normal cells. These microRNAs might be involved in the process of lymphoma transformation. SAM analysis aimed to differentiate GCB-like and ABC-like cell lines identified 11 microRNAs, only 3 of which were present in the cell of origin classifier. This observation suggests that there is also a difference in expression of microRNAs not directly related to the distinct cell of origin between the DLBCL subtypes. In summary, our results demonstrate that the microRNA profile changes during the GC reaction as well as during malignant transformation. Specific microRNAs can regulate key transcription factors controlling the processes of lymphocyte differentiation and transformation.

Constitutive Lymphoid Expression of the Nuclear Form of RNase H1 Is Associated with a Developmental Bottleneck at the Pro-B Cell Stage of B Cell Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4702-4702
Author(s):  
Lina A. Gugliotti ◽  
Kiran B. Sakhuja ◽  
Hongsheng Wang ◽  
Julia Pinkhasov ◽  
Paul E. Love ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Dna Sequences ◽  
Conflicts Of Interest ◽  
Cell Receptor ◽  
Chain Gene ◽  
B Cell Differentiation ◽  
Loop Formation ◽  
Dna Breaks ◽  
Cell Stage

Abstract Abstract 4702 The development of B lymphocytes and the process of lineage determination are initiated by expression of a set of transcriptional regulators leading to V(D)J recombination events initiated by double-strand DNA breaks. Subsequently, these recombinations form DNAs that permit transcription of immunoglobulin genes and translation of the corresponding mRNAs, first by joining the V(D)J DNA sequences, then by recombination, that generates various isotypes of immunoglobulins by class-switch recombination (CSR). Formation of R-loops, regions containing RNA/DNA hybrid and a displaced single-stranded DNA, have been shown to lead to recombination in bacteria, yeast, HeLa and chick cells. Expression in each of these cases of excess ribonuclease H1 (RNase H1), a class of enzymes that degrade RNA in RNA/DNA hybrids, has ameliorated the deleterious effects and decreased recombinational events associated with R-loop formation. R-loops have been observed following transcription of the switch regions that occurs during CSR. The possibility that R-loops are important in V(D)J recombination has not been addressed, and whether ribonucleases H (RNases H) play a role in this process is still uncertain. Transgenic (TG) mice that overexpress RNase H1 in B and T cells (M27F7) were employed in this study. FACS analysis of hematopoietic cells from TG mice revealed a decrease in pre-B cells in the bone marrow. The data indicate a block at the pro-B to pre-B stage of B cell development, which may be the result of apoptosis due to the failure to generate a productive VH-D-JH rearrangement and expression of the pre-B cell receptor. A few B cells that successfully passed these checkpoints predominately differentiated into marginal zone and B1a cells in the peripheral lymphoid organs of the TG mice. These data suggest that R-loops are important in H chain gene rearrangement. This research is supported by the Intramural Research Program of the National Institutes of Health, the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Allergy and Infectious Diseases. Disclosures: No relevant conflicts of interest to declare.

EBV MicroRNA Expression in Virus Driven B-Cell Differentiation and Lymphomagenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 93-93
Author(s):  
Jamie P Nourse ◽  
Pauline Crooks ◽  
Do Nguyen Van ◽  
Kimberley Jones ◽  
Nathan Ross ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Phase 1 ◽  
Differentiation Process ◽  
B Cell Differentiation ◽  
B Cell Lymphomas

Abstract Abstract 93 Lymphomagenesis is a complex process, in part reflecting the nature of the transforming event, as well as the developmental stage of the cell. In the B-cell differentiation represents a continuum that is initiated when a naïve B-cell encounters antigen, undergoes a germinal centre (GC) reaction and ends with terminal differentiation into either a memory or plasma B-cell. Interruption of this process by a transforming event may result in a clonal proliferation where differentiation of the cell is blocked at this stage. The majority of B-cell lymphomas are derived from GC or post-GC B-cells. As physiologically relevant human models that emulate the various stages of B-cell differentiation are lacking we rationalized that in-vitro utilization of the B-cell lymphotrophic Epstein-Barr virus (EBV) would provide insights into this process. In one scenario, EBV infects naïve B-cells and drives a differentiation process paralleling the GC reaction through a well-characterized series of latency gene expression programs. EBV is also implicated in a range of GC and post-GC derived B-cell lymphomas (including Burkitt's, Hodgkin's, PTLD and DLBCL). Using high efficiency EBV infection of isolated naïve B-cells from EBV seronegative subjects, we have demonstrated that EBV infection provides a highly relevant in-vitro model that accurately reflects three distinct phases in the GC differentiation process. Alterations in the expression of a broad range of genes associated with the differentiation of the naïve B-cell were observed within 24 hours of infection and within four days of infection a process exhibiting many similarities to the GC reaction had taken place. These included BCL6, the levels of which were rapidly down-regulated within 24 hours indicating activation of the naïve B-cell. Levels of the memory cell marker CD27 steadily increased over 24 to 96 hours, while BLIMP1 expression increased, peaking at 48 hours. An increase in AID expression over 8 to 48 hours was consistent with somatic hypermutation and isotype switching. Finally a dramatic elevation in expression of the GC associated oncogene LMO2 was observed after two days followed by an equally dramatic downregulation after two weeks. Within two weeks of infection (phase 1), B-cells progressed through a GC-like phase followed by a one week transition state (phase 2) after which continued culture resulted in further differentiation to cells with the phenotypic hallmarks of post-GC cells (phase 3). MicroRNAs (miRNAs) are small non-coding RNAs, which act as negative regulators of gene expression. miRNA expression reflects the developmental lineage and differentiation state of several human cancers and over-expression is implicated in lymphomagenesis. They are also associated with the development of the GC reaction. EBV expresses at least 39 unique miRNAs from the BART and BHRF1 clusters within the viral genome. These EBV miRNAs are differentially expressed in tumour cell lines, suggesting roles during EBV-driven B-cell differentiation and lymphomagenesis. The relationship between EBV miRNAs and the kinetics of EBV driven B-cell differentiation has not been characterized. In our model we find distinct miRNA expression kinetics, coincidental with gene expression changes during B-cell differentiation, suggesting that these regulatory molecules may be involved in the GC process. Although a small number of EBV miRNAs were expressed at low levels early in the GC-like phase 1, the majority were up-regulated during the transition phase 2, exhibiting a subsequent partial down-regulation in the post-GC-like phase 3. The three phases were coincident with differential BART and BHRF1 promoter usage and alternate splicing. Strikingly, application of the infection model to primary patient samples and lymphoma cell-lines revealed that lymphomas clustered within distinct phases, reflecting the full continuum of the B-cell differentiation process. Interestingly, the majority of PTLD samples clustered within the transition phase, whereas Burkitt's and Hodgkin's lymphoma sample segregated with the GC stage. Application of our gene expression and miRNA data to cell-lines and a range of GC and post-GC EBV-positive lymphomas of various histological types indicate that our B-cell differentiation model can be used to accurately classify B-cell lymphomas in a physiologically relevant manner according to the stage of arrested B-cell differentiation. Disclosures: No relevant conflicts of interest to declare.

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