Blimp-1 is a transcriptional repressor able to drive the terminal differentiation of B cells into Ig-secreting plasma cells. We have created mice with a B cell-specific deletion of prdm1, the gene encoding Blimp-1. B cell development and the number of B cells responding to antigen appear to be normal in these mice. However, in response to either TD or TI antigen, serum Ig, short-lived plasma cells, post-GC plasma cells, and plasma cells in a memory response are virtually absent, demonstrating that Blimp-1 is required for plasmacytic differentiation and Ig secretion. In the absence of Blimp-1, CD79b(+)B220(-) pre-plasma memory B cell development is also defective, providing evidence that this subset is an intermediate in plasma cell development. B cells lacking Blimp-1 cannot secrete Ig or induce muS mRNA when stimulated ex vivo. Furthermore, although prdm1-/- B cells fail to induce XBP-1, XBP-1 cannot rescue plasmacytic differentiation without Blimp-1.

(14). BSAP is also important for proliferation and isotype switching in germinal center B cells (4,11,16,20,30). BSAP is expressed throughout B-cell development until the terminally differentiated plasma cell stage (1, 4). BSAP can either activate or repress transcription (33). Targets of BSAP activation include VpreB, 5, CD19, and blk (B lymphoid kinase) (8,15,17,22,35). BSAP represses J chain, the immunoglobulin heavy-chain 3ЈC␣ enhancer and XBP-1 (12,25,28).B-lymphocyte-induced maturation protein 1 (Blimp-1, encoded by the prdm1 gene) is a critical regulator of plasma cell differentiation, induced during cytokine-dependent differentiation of a B-cell lymphoma line (BCL-1) (29) and after lipopolysaccharide (LPS) treatment of primary murine splenocytes (2). Blimp-1 is expressed in all plasma cells and in a subset of germinal center B cells with a partial plasma cell phenotype but not in memory B cells (3). Ectopic expression of Blimp-1 in BCL-1 cells and in primary splenic B cells is sufficient to cause terminal differentiation and immunoglobulin M (IgM) secretion (2,19,26,29).Blimp-1 is a transcriptional repressor. Its DNA-binding activity is conferred by five zinc-finger motifs (7), whereas association with histone deacetylases (34) and hGroucho (24) is required for transcriptional repression. One important target of Blimp-1 repression is c-myc (10). Although repression of c-myc is necessary for terminal differentiation of BCL-1 cells, it is not sufficient, suggesting the existence of additional Blimp-1 targets (9). Indeed, MHC2A, encoding CIITA, a coactivator for major histocompatibility class II (MHC-II) transcription, was recently identified as a Blimp-1 target, providing a mechanism for extinction of MHC-II expression during plasma cell differentiation (19). We demonstrate here that Blimp-1 represses Pax-5 and show that Blimp-1-dependent repression of Pax-5 is required for plasma cell differentiation. MATERIALS AND METHODSCell culture. BCL-1 (CW13.20-3B3, ATCC CRL 1669), P3X (P3X63Ag8), 18-81 Raji, and primary splenocytes were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products, Inc.), 20 g of gentamicin (Gemini)/ml, and 50 M ␤-mercaptoethanol. To induce differentiation of BCL-1, cells (5 ϫ 10 5 cells/ml) were stimulated with interleukin-2 (IL-2) and IL-5, as described previously (29), for various times. 3T3 and Phoenix cells (G. Nolan, Stanford University) were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and 20 g of gentamicin/ ml. WI-L2 transfectants were cultured in the phenol red-free RPMI medium supplemented with 10% charcoal-dextran-treated FBS (HyClone) and penicillin-streptomycin (Gibco-BRL) and cultured in the presence of the selection antibiotic, hygromycin B (500 g/ml; Gibco-BRL). 4-Hydroxytomaxifen was dissolved in 70% ethanol (1 M) and CdSO 4 (5 M) from Sigma.Plasmids. To generate a Blimp-1 binding site mutated reporter, a wild-type luciferase reporter dependent on the Pax-5 promoter (BSAP-Luc) (18) was used as ...

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor that is sufficient to trigger terminal differentiation in the B cell lymphoma BCL-1. In this study, we have determined the expression pattern of Blimp-1 in vivo in primary and secondary lymphoid organs of humans and immunized mice. Blimp-1 is expressed in plasma cells derived from either a T-independent or T-dependent response in plasma cells that have undergone isotype switching and those resulting from secondary immunization. Blimp-1 is also present in long-lived plasma cells residing in the bone marrow. However, Blimp-1 was not detected in memory B cells. This expression pattern provides further evidence of a critical role for Blimp-1 in plasma cell development, supporting earlier studies in cultured lines. Significantly, Blimp-1 was also found in a fraction (4–15%) of germinal center B cells in murine spleen and human tonsils. Blimp-1 expression in the germinal center is associated with an interesting subset of cells with a phenotype intermediate between germinal center B cells and plasma cells. In the mouse, Blimp-1+ germinal center B cells peak at day 12 postimmunization and disappear soon thereafter. They are not apoptotic, some are proliferating, they express germinal center markers peanut agglutinin or CD10 but not Bcl-6, and most express CD138 (syndecan-1), IRF4, and cytoplasmic Ig. Together, these data support a model in which B cell fate decisions occur within the germinal center and Blimp-1 expression is critical for commitment to a plasma cell, rather than a memory cell, fate.

Plasma cells are terminally differentiated final effectors of the humoral immune response. Plasma cells that result from antigen activation of B-1 and marginal zone B cells provide the first, rapid response to antigen. Plasma cells that develop after a germinal center reaction provide higher-affinity antibody and often survive many months in the bone marrow. Transcription factors Bcl-6 and Pax5, which are required for germinal center B cells, block plasmacytic differentiation and repress Blimp-1 and XBP-1, respectively. When Bcl-6-dependent repression of Blimp-1 is relieved, Blimp-1 ensures that plasmacytic development is irreversible by repressing BCL-6 and PAX5. In plasma cells, Blimp-1, XBP-1, IRF4, and other regulators cause cessation of cell cycle, decrease signaling from the B cell receptor and communication with T cells, inhibit isotype switching and somatic hypermutation, downregulate CXCR5, and induce copious immunoglobulin synthesis and secretion. Thus, commitment to plasmacytic differentiation involves inhibition of activities associated with earlier B cell developmental stages as well as expression of the plasma cell phenotype.

Abstract. Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SVinduced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 IxM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Overexpression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SVinduced NF-kappa B activation. To determine if NFkappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.