The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.

The IRF and Ets families of transcription factors regulate the expression of a range of genes involved in immune cell development and function. However, the understanding of the molecular mechanisms of each family member has been limited due to their redundancy and broad effects on multiple lineages of cells. Here, we report that double deletion of floxed Irf8 and Spi1 (encoding PU.1) by Mb1-Cre (designated DKO mice) in the B cell lineage resulted in severe defects in the development of follicular and germinal center (GC) B cells. Class-switch recombination and antibody affinity maturation were also compromised in DKO mice. RNA-seq (sequencing) and ChIP-seq analyses revealed distinct IRF8 and PU.1 target genes in follicular and activated B cells. DKO B cells had diminished expression of target genes vital for maintaining follicular B cell identity and GC development. Moreover, our findings reveal that expression of B-cell lymphoma protein 6 (BCL6), which is critical for development of germinal center B cells, is dependent on IRF8 and PU.1 in vivo, providing a mechanism for the critical role for IRF8 and PU.1 in the development of GC B cells. IRF8 | PU.1 | follicular B cells | BCL6 | germinal center B cell development in the bone marrow (BM) has been wellcharacterized as involving three consecutive stages: (i) B cell lineage specification and commitment at the pre-pro-B cell stage, (ii) pre-B cell receptor (BCR) expression and selection at the pre-B cell stage, and (iii) IgM BCR expression and selection at the immature B cell stage. Several transcription factors are vital for progression through these stages. For example, EBF, E2A, and Pax5 are key regulators for B cell lineage commitment and identity maintenance (1). The IFN regulatory factor (IRF) family members IRF4 and IRF8 and Ets family members PU.1 and SpiB are essential for Ig light-chain gene expression and the generation of immature B cells (2-4). Studies of PU.

SUMMARY:Spontaneous lymphomas occur at high frequency in NFS.V ϩ mice, strains congenic for ecotropic murine leukemia virus (MuLV) proviral genes and expressing virus at high titer. In the present study, a total of 703 NFS.V ϩ lymphomas were studied by histopathology, immunophenotypic analysis, immunoglobulin heavy chain or T cell receptor ␤ chain rearrangements, and somatic ecotropic MuLV integrations; 90% of the lymphomas tested were of B cell lineage. Low-grade tumors included small lymphocytic, follicular, and splenic marginal zone lymphomas, while high-grade tumors comprised diffuse large-cell (centroblastic and immunoblastic types), splenic marginal zone, and lymphoblastic lymphomas. Comparison of mice of similar genetic background except for presence (NFS.V ϩ ) or absence (NFS.V Ϫ ) of functional ecotropic MuLV genomes showed that NFS.V Ϫ clonal lymphomas developed at about one-half the rate of those occurring in NFS.V ϩ mice, and most were low-grade B cell lymphomas with extended latent periods. In NFS.V ϩ mice, clonal outgrowth, defined by Ig gene rearrangements, was associated with acquisition of somatic ecotropic proviral integrations, suggesting that, although generation of B cell clones can be virus independent, ecotropic virus may act to increase the rate of generation of clones and speed their evolution to lymphoma. The mechanism remains undefined, because only rare rearrangements were detected in several cellular loci previously associated with MuLV insertional mutagenesis. (Lab Invest 2000, 80:159-169).O ne of the earliest mammalian animal models in cancer research was the AKR mouse strain developed by Jacob Furth about 70 years ago. Selective breeding yielded mice with nearly 100% mortality due to thymic lymphoma in 7 to 14 months (Cole and Furth, 1941). AKR and the limited number of other T cell lymphoma-prone mouse strains (C58, HRS, and some AKXD recombinant inbred (RI) lines) (Gilbert et al, 1993;Green et al, 1980;MacDowell and Richter, 1935;Meier et al, 1969;Mucenski et al, 1986Mucenski et al, , 1988 have been intensively studied, yielding a partial molecular understanding of the role of ecotropic and recombinant mink cell focus-inducing (MCF) murine leukemia viruses (MuLV) in pathogenesis of this earlyonset lymphoma (Cloyd et al, 1980;Hartley et al, 1977;Holland et al, 1985; M c Grath and Weissman, 1979;Rowe and Hartley, 1983;Stoye et al, 1991;Thomas et al, 1984). In contrast to those of T cell lineage, spontaneous B cell lymphomas are most commonly seen in aged mice of strains in which T cell lymphoma does not predominate. For example, the incidence of B cell lymphoma of various types may be 30% or more in unmanipulated, aged BALB/c, C57BL (Frith and Wiley, 1981), AKR.Fv-1 b (HaranGhera et al, 1993), CWD (Angel and Bedigian, 1984;Mucenski et al, 1988;Thomas et al, 1989), or certain AKXD RI lines (Gilbert et al, 1993;Mucenski et al, 1986) held for up to 3 years, as well as in thymectomized AKR (Peled and Haran-Ghera, 1985) and Emyc transgenic mice (Adams et al, 1985). In SL/Kh (Shimada e...