Aliquots were assayed daily for apoptosis with 7-aminoactinomycin-D (Calbiochem) and analyzed by circulation cytometry, as described (23C25). An additional intracellular fluorescent dye, BODIPY red (Molecular Probes) was used to ascertain cell division in retrovirally transduced B cells. of B cells to antigen and expand the potential contributions of to the genesis of lymphomas. (2, 3). The maintenance of the anergic phenotype requires the continuous activation of the BCR by a low-avidity cognate antigen (4). This state can be accomplished in part by the alteration of the specificity of the BCR through receptor editing (5, 6). The gene encodes a 64-kDa transcription factor that is expressed in many tissues (7). was originally identified as the cellular progenitor of the viral oncogene, has been implicated in many human tumors (8, 9). Prominent among these tumors are PRT062607 HCL diverse forms of lymphoma (10). Accordingly, the normal function of has been shown to have important functions in the development, proliferation, and survival of lymphocytes (11C13). We found that certain transgenes of can elicit a murine lymphoma with many similarities to Burkitt’s lymphoma (BL) (Y.R., J. Duda, K.A.F., and J.M.B., unpublished results). The tumor arises from cooperation between and an autoantigenic stimulus of B cells, which in turn requires a breach of immune tolerance. Here, we demonstrate that this overexpression of itself accounts for the breach of tolerance, and we attribute this effect to the ability of to serve as a surrogate for cytokines. Materials and Methods Mice. Mice transporting the E-transgene have been explained (14). The TRE-and MMTV-rtTA mice have been explained (15, 16). We crossbred these strains to combine the two transgenes in a single strain (MMTV-rtTA/TRE-transgene can be repressed by the administration of tetracycline or doxycycline. We also used BCRHEL mice, which express a rearranged BCR from your endogenous Ig promoter, and soluble hen egg lysozyme (sHEL) mice, which ubiquitously express a transgene for the soluble form of hen egg lysozyme under the control of the metallothionein promoter. These two strains have been explained (17). All transgenic mouse lines were genotyped by PCR as explained (14, 17), except that this E-mouse strain was genotyped by a PCR method using the following primers: E-locus was targeted have been explained (19). Briefly, the ORF with a Pgk-Hprt cassette. The hypomorphic allele (locus. The IL-4C/C mice have been explained (20). All animals were maintained in accordance with the guidelines of the Committee on Animal Research at the University or college of California, San Francisco and the National Research Council. Phenotypic Analysis. The cells present in the lymphoid organs of normal and tumor-bearing mice were immunophenotyped by circulation cytometry. Single-cell suspensions were prepared from your lymph nodes, spleens, thymus, and bone marrow. The cell suspensions were incubated with 1:50 dilutions of antibodies on ice for 30 min, and were then washed in FACS buffer (1% BSA in PBS plus 0.05% sodium azide) and fixed in PBS containing 1% paraformaldehyde. Cells were stained with antibodies to one or more of the following markers: B220, Thy1.2, Mac-1, IgM (pan), IgMa, IgMb, IgD (pan) and IgDa, CD4, CD5, CD8, CD19, CD21, CD23, CD25, CD44, CD62L, CD69, CD80, and CD86 (all obtained from Pharmingen). Hen egg lysozyme (HEL) staining were carried Rabbit Polyclonal to Cytochrome P450 21 out by incubating cell suspensions with 1 mg/ml HEL (Sigma) in FACS PRT062607 HCL buffer. These cells were washed and incubated with Hy9-biotin (HEL-specific monoclonal antibody, a kind gift from J. Cyster, University or college of California, San PRT062607 HCL Francisco), followed by streptavidin-phycoerythrin (PE) (Pharmingen). Serum ELISA. The levels of total serum immunoglobulins were determined by using a capture ELISA, as explained (17). The levels of serum anti-HEL antibodies were determined by performing a solid-phase ELISA for HEL, as explained (17). Blood samples were obtained from mice and allowed to clot by incubating at room heat for 2 h. Samples were centrifuged for 30 min at 5,000 by cultivation with 1 g/ml of anti-CD40 IgM (Pharmingen) and 2 g/ml anti-IgM-F(ab)2 (Jackson ImmunoResearch) for 4 days. Cell division profiles were determined by circulation cytometric analysis of CFSE. For survival assays, B cells that had been activated for 4 days were washed twice and incubated in total media. Aliquots were assayed daily for apoptosis with 7-aminoactinomycin-D (Calbiochem) and analyzed by circulation cytometry, as explained (23C25). An additional intracellular fluorescent dye, BODIPY reddish (Molecular Probes) was used to ascertain cell PRT062607 HCL division in retrovirally transduced B cells. In those experiments, activated B cells that had been retrovirally transduced with GFP-expressing retroviruses were labeled with a final concentration of 10 nM BODIPY reddish, as explained (22). We also measured B cell proliferation as determined by the incorporation of [3H]thymidine, as explained (18). Retroviral Vectors and Lymphocyte Infections. Naive B cells were purified and activated as explained above. These activated B.