Dysregulation of MMSET Is Tumorigenic In-Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 74-74 ◽  
Author(s):  
Marta Chesi ◽  
Kruti Naik ◽  
Davide F. Robbiani ◽  
Maurizio Affer ◽  
Helen D. Nickerson ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Transgenic Mice ◽  
Cell Lines ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Critical Role ◽  
Lymphoid Cells ◽  
Proximal Promoter

Abstract Approximately 15% of multiple myeloma (MM) is characterized by a t(4;14) translocation that causes the simultaneous dysregulation of MMSET on der(4) and fibroblast growth factor receptor 3 gene (FGFR3) on der(14). We reported several lines of evidence indicating a role for FGFR3 in myeloma tumorigenesis. First, activated FGFR3 is an oncogene capable of transforming fibroblasts. Second, FGFR3 activating mutations are acquired by MM cells during tumor progression. Third, targeted inhibition of FGFR3 leads to terminal differentiation and apoptosis in two t(4;14) MM cell lines. However, expression of FGFR3, but never of MMSET, is lost in about 25% of t(4;14) MM. Therefore, the overexpression of MMSET in all MM tumors with a t(4;14) translocation, and its homology to MLL, the oncogene on 11q23 translocated in acute leukemia suggest a critical role for MMSET in MM. To determine whether MMSET is an oncogene in vivo, we have generated transgenic mice in which MMSET expression in driven in lymphocytes by the lck proximal promoter juxtaposed to the Emu enhancer. Using the same expression vector we and others have obtained specific, high levels of transgene expression in B and T cells from spleen, bone marrow and thymus. Four transgenic lines were generated and although we detected MMSET expression in T cells in each of them, unexpectedly no expression in B cells was seen. This is consistent with our inability to ectopically express MMSET in B cell lines. Nevertheless B lymphoid tumors expressing MMSET developed at 23 month of age in each line (18/51 mice). Only 1/19 wild type matching control mice developed a splenomegaly. By Southern blot, monoclonal rearrangements of IgH, IgL and TCR β were detected within the same tumor population. In conclusion, this is the first report that MMSET is an oncogene capable of transforming lymphoid cells in an animal model. We are currently crossing these mice with FGFR3 transgenic mice to assess cooperation between these two oncogenes in tumorigenesis. Obviously a more restricted expression of MMSET in germinal center cells is required to investigate the role of MMSET in MM. Therefore, as we have done for c-myc, we are generating new transgenic mice in which MMSET expression will be activated sporadically in germinal center B cells by somatic hypermutation.

scholarly journals The effect of in vivo IL-7 deprivation on T cell maturation.

1995 ◽  
Vol 181 (4) ◽  
pp. 1399-1409 ◽  
Author(s):  
S K Bhatia ◽  
L T Tygrett ◽  
K H Grabstein ◽  
T J Waldschmidt
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Present Report ◽  
Critical Role ◽  
Cell Maturation ◽  
Double Negative ◽  
Heat Stable Antigen ◽  
Single Positive

A number of previous studies have suggested a key role for interleukin 7 (IL-7) in the maturation of T lymphocytes. To better assess the function of IL-7 in lymphopoiesis, we have deprived mice of IL-7 in vivo by long-term administration of a neutralizing anti-IL-7 antibody. In a previous report (Grabstein, K. H., T. J. Waldschmidt, F. D. Finkelman, B. W. Hess, A. R. Alpert, N. E. Boiani, A. E. Namen, and P. J. Morrissey. 1993. J. Exp. Med. 178:257-264), we used this system to demonstrate the critical role of IL-7 in B cell maturation. After a brief period of anti-IL-7 treatment, most of the pro-B cells and all of the pre-B and immature B cells were depleted from the bone marrow. In the present report, we have injected anti-IL-7 antibody for periods of up to 12 wk to determine the effect of in vivo IL-7 deprivation on the thymus. The results demonstrate a > 99% reduction in thymic cellularity after extended periods of antibody administration. Examination of thymic CD4- and CD8- defined subsets revealed that, on a proportional basis, the CD4+, CD8+ subset was most depleted, the CD4 and CD8 single positive cells remained essentially unchanged, and the CD4-, CD8- compartment actually increased to approximately 50% of the thymus. Further examination of the double negative thymocytes demonstrated that IL-7 deprivation did, indeed, deplete the CD3-, CD4-, CD8- precursors, with expansion of this subset being interupted at the CD44+, CD25+ stage. The proportional increase in the CD4-, CD8- compartment was found to be due to an accumulation of CD3+, T cell receptor alpha, beta + double negative T cells. Additional analysis revealed that anti-IL-7 treatment suppressed the audition/selection process of T cells, as shown by a significant reduction of single positive cells expressing CD69 and heat stable antigen. Finally, the effects of IL-7 deprivation on the thymus were found to be reversible, with a normal pattern of thymic subsets returning 4 wk after cessation of treatment. The present results thus indicate a central role for IL-7 in the maturation of thymic-derived T cells.

Transformation of BCR-deficient germinal-center B cells by EBV supports a major role of the virus in the pathogenesis of Hodgkin and posttransplantation lymphomas

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4345-4350 ◽  
Author(s):  
Dörte Bechtel ◽  
Julia Kurth ◽  
Claus Unkel ◽  
Ralf Küppers
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Lymphoproliferative Disease ◽  
Barr Virus ◽  
Classic Hodgkin Lymphoma ◽  
Epstein Barr

In classic Hodgkin lymphoma (HL) and posttransplantation lymphoproliferative disease (PTLD), 2 malignancies frequently associated with Epstein-Barr virus (EBV), the tumor cells often appear to derive from B-cell receptor (BCR)–deficient and therefore preapoptotic germinal center (GC) B cells. To test whether EBV can rescue BCR-less GC B cells, we infected human tonsillar CD77+ GC B cells in vitro with EBV. More than 60 monoclonal lymphoblastoid cell lines (LCLs) were established. Among these, 28 cell lines did not express surface immunoglobulin (sIg). Two of the sIg-negative cell lines carry obviously destructive mutations that have been introduced into originally functional VH gene rearrangements during the process of somatic hypermutation. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) showed that in most other lines the sIg deficiency was not simply the result of transcriptional down-regulation, but it was rather due to posttranscriptional defects. These findings strongly support the idea that EBV plays a central role in the pathogenesis of classic HL and PTLD by rescuing BCR-deficient, preapoptotic GC B cells from apoptosis, and that EBV infection renders the cells independent from survival signals normally supplied by a BCR. The monoclonal LCLs represent valuable models for early stages of lymphoma development in classic HL and PTLD.

VavP-Bcl2 transgenic mice develop follicular lymphoma preceded by germinal center hyperplasia

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2276-2283 ◽  
Author(s):  
Alexander Egle ◽  
Alan W. Harris ◽  
Mary L. Bath ◽  
Lorraine O'Reilly ◽  
Suzanne Cory
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Follicular Lymphoma ◽  
Germinal Center ◽  
Cd4 T Cells ◽  
Chromosome Translocation ◽  
Regulatory Sequences ◽  
Germinal Center Hyperplasia ◽  
B Lymphoid

Abstract In human follicular lymphoma the t(14; 18) chromosome translocation activates the antiapoptotic oncogene Bcl2 by linking it to the immunoglobulin heavy chain (IGH) locus. Transgenic mice expressing Bcl2 controlled by an Igh enhancer (Eμ) do not develop follicular lymphoma, although they do have an increased incidence of other B-lymphoid neoplasms. We have now analyzed tumorigenesis in mice bearing a Bcl2 transgene controlled by Vav gene regulatory sequences (VavP), which confer expression in multiple hematopoietic lineages. Unlike Eμ-Bcl2 mice, many VavP-Bcl2 mice older than 10 months developed follicular lymphoma. Young VavP-Bcl2 mice had an overabundance of enlarged germinal centers and greatly elevated numbers of cycling B cells that had undergone IgH class switching and V-gene hypermutation. The peripheral T-cell compartment was larger in the VavP-Bcl2 mice than in Eμ-Bcl2 strains and, notably, CD4 T cells were 5-fold increased over normal. The germinal center hyperplasia required CD4 T cells, because it could be abolished by anti-CD4 antibody in vivo. VavP-Bcl2 mice also had a propensity to develop kidney disease of the autoimmune type. We suggest that the increased survival capacity of B and T cells fosters prolonged germinal center reactions, and that autoreactivity and hypermutation conspire to generate follicular lymphoma.

Impaired germinal center formation and recall T-cell–dependent immune responses in mice lacking the costimulatory ligand B7-H2

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1381-1388 ◽  
Author(s):  
Siew-Cheng Wong ◽  
Edwin Oh ◽  
Chee-Hoe Ng ◽  
Kong-Peng Lam
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
Heavy Chain ◽  
Germinal Center ◽  
Critical Role ◽  
Mutant Mice ◽  
Type Ii ◽  
Germinal Center Formation

Abstract B7-H2, which is expressed constitutively on B cells and binds the inducible costimulator (ICOS) on antigen-activated T cells, is a member of the B7 family of costimulatory ligands. We have inactivated B7-H2 in the mouse. B7-H2–/– mice generate normal populations of B and T cells in their various lymphoid organs but have lower basal levels of heavy chain class–switched antibodies in their sera. These mice are able to mount normal immune responses to both type I and type II T-cell–independent antigens. However, their pattern of responses to a T-cell–dependent antigen is altered, with greatly reduced production of antigen-specific heavy chain class–switched antibodies, the levels of which could not be elevated even with repeated immunizations. This suggests a critical role for B7-H2 in the recall phases of the immune response. Germinal center formation is also impaired in the mutant mice. While B cells from the mutant mice could response normally to anti-IgM, anti-CD40, and lipopolysaccharide stimulation, the production of T-helper–type II cytokines such as interleukin-4 (IL-4) and IL-10 by primed CD4+ T cells from mutant mice were reduced. This indicated that the defects in humoral responses and germinal center formation in B7-H2–deficient mice are due to the lack of T-cell–mediated help to the B cells. Hence, B7-H2 on B cells is important for recruiting T-cell help via its interaction with ICOS and plays a critical role in costimulating humoral immune responses.

scholarly journals Chronic GvHD Is Characterized By Impaired B Cell Development in the Bone Marrow

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1883-1883
Author(s):  
Oleg Kolupaev ◽  
Michelle West ◽  
Bruce R. Blazar ◽  
Stephen Tilley ◽  
James Coghill ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
B Lymphopoiesis

Abstract Background. Chronic-graft-versus-host disease (cGvHD) continues to be a major complication following allogeneic hematopoietic stem cell transplantation (HSCT). Despite significant progress, mechanisms underlying development of the pathology are yet to be fully understood. Recent studies utilizing mouse models and patient samples have demonstrated a critical role for B cells in GvHD pathogenesis. Bone marrow (BM)-derived B cells can produce auto-reactive antibodies causing tissue fibrosis and multiorgan cGvHD. Impaired B cell homeostasis in the periphery, activation due to abnormally high levels of B cell-activating factor (BAFF), increased survival of auto-reactive B cells and aberrant BCR signaling are shown to be important for disease progression in cGvHD patients. Murine models also highlighted the critical role of germinal center reactions, particularly interactions between T follicular helper (Tfh) cells and B cells for generation of auto-antibodies which are responsible for triggering immune responses and cell-mediated toxicity. A growing body of evidence has emerged highlighting the fact that BM itself is a target organ during acute GvHD (aGvHD) with recent work suggesting a role for donor CD4+ T cells in BM specific aGvHD. Our group has shown that patients with higher numbers of BM B cell precursors were less likely to develop cGvHD after allogeneic HSCT (Fedoriw et al., 2012). These observations indicate clinical relevance of impaired BM B lymphopoiesis for cGvHD development. Methods. In order to investigate the effect of cGvHD on BM B cell development, we used the well-characterized major mismatch B6 into B10.BR model of systemic cGvHD. Recipient mice were treated with cyclophosphamide on day -3 and -2, irradiated with 700 cGy on day -1, and injected with 107 T cell depleted (TCD) BM with or without total splenic T cells (0.5-1x105). Mice were monitored for 30 days, and BM and spleen was harvested and analyzed using flow cytometry. Results. Consistent with patient data, we observed a decrease in the frequency and number of donor-derived uncommitted common lymphoid progenitors (CLP) and B cell progenitors in the BM+ allogeneic T cells group (CLP: 0.17±0.03% vs. 0.06±0.01%, p <0.01; pro B: 2.2 ± 0.5% vs. 0.7 ± 0.3%, p<0.05; pre B: 15.3±1.8% vs. 6.3±2.4%, p<0.05; immature B cells: 5.7±0.7% vs. 2.1±0.7%, p<0.01) (Fig.1). As previously reported for this model, we also found a decrease in the frequency of follicular (FO) B cells (Flynn et al., 2014). We hypothesized that during cGvHD the B cell progenitor BM niche is affected by donor CD4+ T cells leading to impaired B lymphopoiesis. Bone marrow from BM+T cell animals had a significantly higher frequency of CD4+ cells compared to the control group (0.45±0.06% vs. 0.2±0.02%). Depletion of CD4+ T cells using anti-CD4 antibody during the first two weeks after transplant improved pathology scores and prevented weight loss in BM+T cells mice. We also observedpartial recovery of B cell progenitors and Lin-CD45-CD31-CD51+ osteoblasts (OB) in animals treated with anti-CD4 antibodies (pre B 3.5±1.1% vs. 20.4±4.5%, p<0.05; immature B: 1.9±0.9% vs. 3.5±0.3%; OB: 0.8±0.1% vs.1.2±0.2%). A recent study showed that activation and proliferation of conventional T cells in aGvHD model can be prevented by in vivo expansion of regulatory T cells (Tregs) using αDR3 antibody (4C12). We adopted this approach to determine whether Tregs can suppress the cytotoxic effect of donor CD4+ T cells in BM in cGvHD model. Animals that received T cells from 4C12-treated donors had an increase in survival and lower cGvHD pathology scores. These mice also had higher frequency of pro B, pre B, and immature B cells compared to the mice infused with T cells from isotype-treated donors. Conclusions. These studies demonstrate that BM development of B lymphocytes is impaired in a mouse model of systemic cGvHD. Our data suggests that donor-derived CD4+ T cells are involved in the destruction of hematopoietic niches in BM, particularly OB, which support B lymphopoiesis. Moreover, depletion of CD4+ T cells and infusion with in vivo expanded Tregs reduced the severity of cGvHD. Thus, Treg therapy in patients with cGvHD may be important for BM B cell development, and improvement of clinical outcomes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Unbalanced Expression of Bcl-2 Family Proteins in Follicular Lymphoma: Contribution of CD40 Signaling in Promoting Survival

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 244-251 ◽  
Author(s):  
Paolo Ghia ◽  
Vassiliki A. Boussiotis ◽  
Joachim L. Schultze ◽  
Angelo A. Cardoso ◽  
David M. Dorfman ◽  
...  
Keyword(s):  
B Cells ◽  
Follicular Lymphoma ◽  
Germinal Center ◽  
Conventional Treatment ◽  
Clinical Course ◽  
Critical Role ◽  
Relative Resistance ◽  
Constitutive Overexpression

Abstract Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.

scholarly journals Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells.

1994 ◽  
Vol 179 (2) ◽  
pp. 425-438 ◽  
Author(s):  
M P Cooke ◽  
A W Heath ◽  
K M Shokat ◽  
Y Zeng ◽  
F D Finkelman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Transgenic Mice ◽  
B Lymphocytes ◽  
Molecular Mechanisms ◽  
Interleukin 4 ◽  
Th Cells

The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.

Enforced BCL6 Expression Inhibits B Cell Development in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1535-1535
Author(s):  
Davide F. Robbiani ◽  
Kaity Colon ◽  
Kruti Naik ◽  
Helen Nickerson ◽  
Maurizio Affer ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Light Chain ◽  
Germinal Center ◽  
Regulatory Elements ◽  
Cell Development ◽  
B Cell Development ◽  
Kappa Light Chain

Abstract The B-Cell Lymphoma 6 (BCL6) gene encodes for a zinc finger motifs containing transcriptional repressor that is frequently dysregulated by chromosomal translocations in germinal center lymphomas. A putative protooncogene, its transforming ability in vivo was reported in I-mu-HA-BCL6 knock-in mice by Cattoretti et al last year. We also tested this assumption in transgenic mice expressing BCL6 in B cells under the control of kappa light chain regulatory elements. We replaced the murine C-kappa locus with the 16kb human BCL6 genomic locus in a construct containing the murine kappa light chain regulatory elements (Vk, EiK, 3′RR). While control transgenics were readily obtained (5/32 founders), only 3/68 founders were positive for the BCL6 transgene, of which only one (bearing a single copy of the transgene) was able to transmit the transgene to its progeny, thus suggesting embryonal toxicity of exogenous BCL6. In the bone marrow, flow cytometry revealed a nearly complete block of B cell development at the pro-B to pre-B transition. This was also the stage at which we first detected expression of EGFP in control reporter mice that were generated in parallel. Spleens of transgenic mice weighed about 50% of control spleens and less than 5% of splenocytes were CD19+ B cells. These were IgM high, IgD intermediate, corresponding to an immature B cell phenotype. Lymph nodes were smaller and B cells barely detected. Peyers’ patches were not visible. Combined, our analysis of 6–8 weeks old VkHABCL6 transgenic mice reveals that enforced expression of BCL6 early in development results in a profound block of B lymphocyte differentiation. How transgenic BCL6 modulates this effect at the transcriptional level remains to be investigated. To test the oncogenic potential of BCL6 in B cells, it will be interesting to precisely turn on this gene in the germinal center.

scholarly journals Development of Mouse Embryonic Stem (ES) Cells: IV Differentiation to Mature T and B Lymphocytes after Implantation of Embryoid Bodies Into Nude Mice

1995 ◽  
Vol 4 (2) ◽  
pp. 79-84 ◽  
Author(s):  
Una Chen ◽  
Hoyan Mok
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nude Mice ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Lymphoid Cells

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combinedin vitroandin vivosystem are discussed.

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