Intrinsic B-cell hyporesponsiveness accounts for self-tolerance in lysozyme/anti-lysozyme double-transgenic mice.

Abstract Abstract 2968 Poster Board II-944 Previously, we identified and validated PIM1 as a differentially expressed gene in mantle cell lymphoma (MCL) patient samples. Further, we have shown PIM1 to be a significant prognostic biomarker in MCL. PIM1 is a serine/threonine kinase that is transcriptionally regulated by cytokines, mitogens, and numerous growth factors. PIM1 cooperates with other oncogenes in tumorigenesis and has been implicated in the development of leukemias, lymphomas, late progression events, and most recently in prostate cancer. PIM1 is overexpressed in aggressive lymphomas, such as the blastoid variant of MCL, and the ABC-subtype of diffuse large B-cell lymphomas (DLBCL). Here, we tested the in vivo cooperation of PIM1 with TCL1 in murine lymphomagenesis by producing double transgenic murine strains. PIM1 transgenic mice overexpress murine PIM1 under the control of the immunoglobulin enhancer Eμ. TCL1 transgenic mice (pEμ-B29-TCL1) fail to down-regulate TCL1 expression in mature B and T cells and provide a unique model for mature B-cell malignancies. We hypothesized that PIM1 would either accelerate TCL1-driven lymphomagenesis, result in the development of immature lymphomas, or both. Lymphoid malignancies were examined by immunohistochemistry and classified by a hematopathologist according to “Mouse Models of Human Blood Cancers” (Li et al., 2008). A Kaplan-Meier plot demonstrated statistically significant acceleration of lymphomagenesis in the PIM1/TCL1 transgenic mice when compared with the single transgenic strains. The median lymphoma-free survival for TCL1 single transgenic mice, PIM1 single transgenic mice, or PIM1/TCL double transgenic mice were 10.0 months, 16.0 months, and 8.5 months, respectively. The results were statistically significant: TCL1 vs. PIM1/TCL1 (p=0.0008), PIM1 vs. PIM1/TCL1 (p<0.0001). PIM1 transgenic mice developed rare [1 of 31 (3.2%)] and early (< 7 months of age) T-cell lymphoblastic lymphomas and more commonly developed late (>12 months of age) B-cell lymphomas. The most common lymphomas in PIM1 single transgenic were low-grade B-cell lymphomas [12 of 31 (38.7%)], mainly follicular lymphomas. A minority of PIM1 single transgenic mice developed aggressive lymphomas [6 of 31 (19.4%)], including DLBCL and Burkitt's lymphoma. In contrast, the majority of TCL1 transgenic mice developed aggressive B-cell lymphomas [21 of 36 (58.3%)], mainly DLBCL, lymphohistiocytic subtype. The majority of PIM1/TCL1 double transgenic mice also developed aggressive B-cell lymphomas [20 of 34 (58.8%)], mainly DLBCL, lymphohistiocytic subtype. The low-grade lymphomas that developed in PIM1/TCL1 mice included 5 cases of lymphoplasmacytic lymphoma (LPL); one of these cases had transformed in addition to a DLBCL. Further, endogenous expression of PIM kinase family members was investigated in a human lymphoma cell line bank (n=40) by quantitative real-time PCR. PIM1, PIM2, and PIM3 were found to be overexpressed in cell lines derived from human lymphoid malignancies of multiple histologies. In summary, aberrant PIM1 overexpression in TCL1 transgenic mice accelerated the development of mature, aggressive B-cell lymphomas. The classification of lymphomas in PIM1/TCL1 mice revealed similar histologies as in TCL1 single transgenic mice, mainly DLBCL. Single transgenic PIM1 mice developed low-grade B-cell lymphomas after prolonged observation time. The expression of all 3 PIM kinase family members in human lymphomas implies that pan-PIM kinase inhibitors should be developed as a potential mechanism of drug resistance to more restricted PIM inhibitors could be compensatory overexpression of the non-targeted Pim family members. A clinical trial with a pan-PIM inhibitor is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

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Mutated EZH2 Collaborates with Myc in Inducing Lymphoma in a Mouse Model

Blood ◽

10.1182/blood.v118.21.227.227 ◽

2011 ◽

Vol 118 (21) ◽

pp. 227-227 ◽

Cited By ~ 1

Author(s):

Tobias Berg ◽

Damian Yap ◽

Silvia Thoene ◽

Tracee Wee ◽

Nathalie Schoeler ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

B Cell ◽

B Cell Lymphoma ◽

S Phase ◽

Brdu Incorporation ◽

Cell Phenotype ◽

Global Changes ◽

Mutant Form ◽

Double Transgenic Mice

Abstract Abstract 227 The histone methyl-transferase EZH2 is frequently mutated in germinal-center derived diffuse-large B cell lymphoma (DLBCL) and follicular lymphoma. We and others have demonstrated that these mutations in tyrosine 641 of EZH2 represent a dominant gain-of-function, enhancing the capability to trimethylate H3K27 in combination with wildtype activity (Yap et al, Blood 2011; Sneeringer et al, PNAS 2010). To further test if these EZH2 mutations can be driver mutations in lymphomagenesis we have now generated and characterized a mouse line transgenic for EZH2 Y641F (the most commonly observed mutant form). Transgenic mice were created on the C57/Bl6 background by pronuclear microinjection of a construct where EZH2 Y641F is expressed from a lymphoid specific Emu-lck promoter. Spleen cells isolated from the transgenic mice displayed a global increase in H3K27me3 when analyzed by Western Blot. However none of the mice showed signs of lymphoma even when observed for prolonged periods of time. As EZH2 mutations often coincide with other mutations in lymphoma, we also combined the expression of EZH2 Y641F with the over-expression of Myc by crossing one of the transgenic lines with Emu-myc transgenic mice. In this combination model, we observed a dramatic acceleration of lymphoma development with the combination of Myc and EZH2 Y641F as compared to Myc alone (Myc alone (n=26) 137.5 days, Myc+EZH2 Y641F (n=20) 51 days, p < 0.0001 Log-Rank (Mantel-Cox) Test). The resulting lymphomas are characterized by a generalized massive lymphadenopathy, splenomegaly (spleen weight 0.416 +/− 0.133 g), bone marrow infiltration (72 +/− 8 %) and lymphocytosis (WBC 138.5 +/− 77.1 ×1000/μl). They show histological features of high-grade disease, and initiate disease in secondary recipients. In contrast to lymphomas observed with Emu-myc alone, all of the lymphomas observed in mice transgenic for both, Myc and EZH2 Y641F, presented with a mature B cell phenotype (B220+IgM+). This shift in differentiation can also be observed in the bone marrow even before development of overt lymphomas. While Myc transgenics mainly show an increase in the B220+IgM−compartment, this is shifted towards B220+IgM+ in double transgenics. Cell cycle analysis by in vivo Brdu incorporation before disease onset shows a drastic increase in the fraction of cells in cycle in the splenic B cells of double transgenic mice (43.3 and 44.9 % cells in S phase) as compared to mice transgenic for Myc alone (10.6 % and 14.2 % cells in S phase). Southern Blot analysis using a probe specific for JH4 showed more than one clonal rearrangement in a majority of the lymphomas in the double-transgenic mice. In summary, EZH2 Y641F can collaborate with Emu-myc in high efficiency lymphoma induction. This new murine lymphoma model now provides a powerful tool to study global changes in the epigenome caused by this frequent mutation and may be a useful system for testing novel treatments. Disclosures: No relevant conflicts of interest to declare.

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A transgenic model of autoimmune hemolytic anemia.

Journal of Experimental Medicine ◽

10.1084/jem.175.1.71 ◽

1992 ◽

Vol 175 (1) ◽

pp. 71-79 ◽

Cited By ~ 166

Author(s):

M Okamoto ◽

M Murakami ◽

A Shimizu ◽

S Ozaki ◽

T Tsubata ◽

...

Keyword(s):

B Cells ◽

Transgenic Mice ◽

Disease Model ◽

Single Chain ◽

Relative Contribution ◽

Treatment And Prevention ◽

Self Tolerance ◽

Genes Encoding ◽

Individual Mouse ◽

Double Transgenic Mice

We made double transgenic mice bearing immunoglobulin heavy and light chain genes encoding an autoantibody against the mouse erythrocyte by the cross of C57BL/6 mice carrying the transgene for each chain of the immunoglobulin. Although no obvious disorders were found in the single-chain transgenic mice, severely anemic symptoms were found in some of the double transgenic mice, in which most B cells express, at least on their surface, the autoantibody reactive to self-antigens on the erythrocyte. Individual double-transgenic mice showed a wide variation of phenotypes between severe anemia and no symptoms. Both deletion and anergy of autoreactive B cells were seen in each individual mouse, but their relative contribution to self-tolerance was variable and not directly related to the severity of anemia or the amount of the autoantibody produced. This transgenic system provides a good autoimmune disease model for exploring its onset mechanism, and means of its treatment and prevention.

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B Lymphocytes Producing Demyelinating Autoantibodies: Development and Function in Gene-targeted Transgenic Mice

Journal of Experimental Medicine ◽

10.1084/jem.188.1.169 ◽

1998 ◽

Vol 188 (1) ◽

pp. 169-180 ◽

Cited By ~ 163

Author(s):

Tobias Litzenburger ◽

Reinhard Fässler ◽

Jan Bauer ◽

Hans Lassmann ◽

Christopher Linington ◽

...

Keyword(s):

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Cell Population ◽

Plasma Cells ◽

Autoimmune Encephalitis ◽

Normal Numbers ◽

Self Tolerance ◽

Normal Differentiation ◽

And Function

We studied the cellular basis of self tolerance of B cells specific for brain autoantigens using transgenic mice engineered to produce high titers of autoantibodies against the myelin oligodendrocyte glycoprotein (MOG), a surface component of central nervous system myelin. We generated “knock-in” mice by replacing the germline JH locus with the rearranged immunoglobulin (Ig) H chain variable (V) gene of a pathogenic MOG-specific monoclonal antibody. In the transgenic mice, conventional B cells reach normal numbers in bone marrow and periphery and express exclusively transgenic H chains, resulting in high titers of MOG-specific serum Igs. Additionally, about one third of transgenic B cells bind MOG, thus demonstrating the absence of active tolerization. Furthermore, peritoneal B-1 lymphocytes are strongly depleted. Upon immunization with MOG, the mature transgenic B cell population undergoes normal differentiation to plasma cells secreting MOG-specific IgG antibodies, during which both Ig isotype switching and somatic mutation occur. In naive transgenic mice, the presence of this substantial autoreactive B cell population is benign, and the mice fail to develop either spontaneous neurological disease or pathological evidence of demyelination. However, the presence of the transgene both accelerates and exacerbates experimental autoimmune encephalitis, irrespective of the identity of the initial autoimmune insult.

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CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation.

Journal of Experimental Medicine ◽

10.1084/jem.183.4.1707 ◽

1996 ◽

Vol 183 (4) ◽

pp. 1707-1718 ◽

Cited By ~ 269

Author(s):

K F Byth ◽

L A Conroy ◽

S Howlett ◽

A J Smith ◽

J May ◽

...

Keyword(s):

Signal Transduction ◽

T Cell ◽

B Cells ◽

Transgenic Mice ◽

B Cell ◽

T Cell Development ◽

Cell Development ◽

Cross Linking ◽

Thymocyte Development ◽

Double Positive

The CD45 transmembrane glycoprotein has been shown to be a protein phosphotyrosine phosphatase and to be important in signal transduction in T and B lymphocytes. We have employed gene targeting to create a strain of transgenic mice that completely lacks expression of all isoforms of CD45. The spleens from CD45-null mice contain approximately twice the number of B cells and one fifth the number of T cells found in normal controls. The increase in B cell numbers is due to the specific expansion of two B cell subpopulations that express high levels of immunoglobulin (IgM) staining. T cell development is significantly inhibited in CD45-null animals at two distinct stages. The efficiency of the development of CD4-CD8- thymocytes into CD4+ CD8+ thymocytes is reduced by twofold, subsequently the frequency of successful maturation of the double positive population into mature, single positive thymocytes is reduced by a further four- to fivefold. In addition, we demonstrate that CD45-null thymocytes are severely impaired in their apoptotic response to cross-linking signals via T cell receptor (TCR) in fetal thymic organ culture. In contrast, apoptosis can be induced normally in CD45-null thymocytes by non-TCR-mediated signals. Since both positive and negative selection require signals through the TCR complex, these findings suggest that CD45 is an important regulator of signal transduction via the TCR complex at multiple stages of T cell development. CD45 is absolutely required for the transmission of mitogenic signals via IgM and IgD. By contrast, CD45-null B cells proliferate as well as wild-type cells to CD40-mediated signals. The proliferation of B cells in response to CD38 cross-linking is significantly reduced but not abolished by the CD45-null mutation. We conclude that CD45 is not required at any stage during the generation of mature peripheral B cells, however its loss reveals a previously unrecognized role for CD45 in the regulation of certain subpopulations of B cells.

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Modulation of T-Cell Functions in KIR2DL3 (CD158b) Transgenic Mice

Blood ◽

10.1182/blood.v94.7.2396.419k17_2396_2402 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2396-2402 ◽

Cited By ~ 26

Author(s):

Anna Cambiaggi ◽

Sylvie Darche ◽

Sophie Guia ◽

Philippe Kourilsky ◽

Jean-Pierre Abastado ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Activation ◽

Cell Activation ◽

Killer Cell ◽

In Vitro Proliferation ◽

Cell Functions ◽

Double Transgenic Mice

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR+ β and γδ T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 × Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.

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