Pax5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. To better understand the functional importance of Pax5 at the later stages of B cell differentiation, we established a Pax5-deficient DT40 B cell line. The Pax5(-/-) cells exhibited slower growth, decreased surface IgM expression, and total loss of B cell receptor signaling. Moreover, the expression of the plasma cell-characteristic transcription factors Blimp-1 and XBP-1 were significantly upregulated and the expression of Bcl-6 diminished in the Pax5(-/-) cells, and this alteration was normalized by restored Pax5 expression. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results indicate that downregulation of Pax5 function promotes the plasma cell differentiation of B cells.

The transcription factor Bcl6 regulates germinal center formation and differentiation of B cells into high-affinity antibody-producing plasma cells. The direct double-negative regulatory circuit between Bcl6 and Blimp-1 is well established. We now reveal alternative mechanisms for Bcl6-mediated regulation of B-cell differentiation to plasma cells and show with DT40 cells that Bcl6 directly promotes the expression of Bach2, a known suppressor of Blimp-1. Moreover, Bcl6 suppresses Blimp-1 expression through direct binding to the IRF4 gene, as well as by promoting the expression of MITF, a known suppressor of IRF4. We also provide evidence that Bcl6 is needed for the expression of AID and UNG, the indispensable proteins for somatic hypermutation and class-switch recombination, and UNG appears to be a direct Bcl6 target. Our findings reveal a complex regulatory network in which Bcl6 acts as a key element dictating the transition of DT40 B cells to plasma cells. Supporting Information available online See accompanying Commentary by Tarlinton IntroductionThe transition of activated B cells into high-affinity antibodyproducing plasmablasts, plasma cells and memory B cells in the germinal centers (GCs) is a biologically unique process involving rapid B-cell proliferation coupled with a high rate of mutation in the variable regions of immunoglobulin (Ig) genes. Considering that the majority of B-cell lymphomas originate from the GC B cells [1], a comprehensive understanding of the transcriptional regulation controlling the transition of B cells to plasma cells is essential. The transcription factors Blimp-1, Xbp-1 and IRF4 have been identified to be critical regulators promoting the Ig secretion and plasma cell phenotype, whereas Pax5, Bcl6, MITF and Bach2 are the known inhibitors of plasma cell development [2].Previous studies have shown that the downregulation of B-cell identity factor Pax5 occurs before the induction of the plasma cell transcription program [3,4], and that the loss of Pax5 expression [4] leads to the downregulation of BCL6. Furthermore, B-cell receptor (BCR) signaling of sufficient affinity leads to rapid phosphorylation and degradation of Bcl6 protein [5], while Bcl6 can also be functionally inactivated by acetylation [6]. These features of Bcl6 make it a prominent molecular trigger that controls the plasma cell differentiation.The transcription factor Bcl6 has been demonstrated to be essential for GC B-cell development, as Bcl6 knockout mice fail to develop GCs and do not produce high-affinity antibodies [7][8][9]. Recent work revealed that Bcl6 is also needed for follicular T helper cell development [10][11][12]. Analyses of Bcl6 target genes in B cells indicate that suppression of apoptosis and cell cycle arrest responses occur through p53, p21 and CHEK1 and Bcl6 also regulates DNA damage responses by controlling ATR [13][14][15][16]. Thus, Bcl6 appears to function as a factor permitting rapid B-cell 2404proliferation and tolerance for DNA damage in GCs. Bcl6 also represses genes that are involve...

The transcription factor Ikaros, a key regulator of hematopoiesis, has an essential role in lymphocyte development. In mice, fetal lymphoid differentiation is blocked in the absence of Ikaros, and whereas T cells develop postnatally, B cells are totally absent. The significance of Ikaros in the B cell development is evident, but how Ikaros regulates B cell function has neither been established nor previously been studied with B cells that lack Ikaros expression. Here we show that disruption of Ikaros in the chicken B cell line DT40 induces a B cell receptor (BCR) signaling defect with reduced phospholipase Cc2 phosphorylation and impaired intracellular calcium mobilization, which is restored by Ikaros reintroduction. Furthermore, we show that lack of Ikaros induces hyperphosphorylation of Casitas B lymphoma protein subsequent to BCR activation. These results indicate that the absolute need of Ikaros for development, cell fate decisions and maintenance of B cells is due to the enhancement of BCR signaling.

Paired box protein 5 (Pax5) is essential for early B cell commitment as well as for B cell development, and continuous expression of Pax5 is required throughout the B cell lineage to maintain the functional identity of B cells. During B cell activation, Pax5 is downregulated before terminal differentiation into antibody‐secreting plasma cells, and enforced expression of Pax5 prevents plasmacytic development. Recently, loss of Pax5 was shown to result in the substantial transition to a plasma cell state, demonstrating a functionally significant role for Pax5 in the regulation of terminal B cell differentiation. Here we elucidate the current understanding about the function of Pax5 as a key inhibitor of plasma cell differentiation.

The Ikaros family transcription factor Aiolos is important for B cell function, since B cells of Aiolos‐null mutant mice exhibit an activated phenotype, enhanced B‐cell receptor (BCR) signalling response and develop a systemic lupus erythematosus (SLE) type autoimmune disease. Aiolos has also been reported to interact with anti‐apoptotic Bcl‐2 and Bcl‐xL in T cells, but whether Aiolos regulates cell death has not been studied in B cells. Here we show that the disruption of Aiolos in the DT40 B cell line induces a cell death sensitive phenotype, as the Aiolos−/− cells are more prone to apoptosis by nutritional stress, BCR cross‐linking, UV‐ or γ‐irradiation. Furthermore, the Aiolos−/− cells have defective Ig gene conversion providing evidence that Aiolos is needed for the somatic diversification of the BCR repertoire. The re‐expression of DNA‐binding isoform Aio‐1 was able to restore the gene conversion defect of the Aiolos‐deficient cells, whereas the introduction of dominant negative isofom Aio‐2 had no effect on gene conversion, thus demonstrating the functional importance of alternative splicing within Ikaros family. Although the Aiolos−/− cells exhibit reduced expression of activation‐induced cytidine deaminase (AID), ectopic AID overexpression did not restore the gene conversion defect in the Aiolos−/− cells. Our findings indicate that Aiolos may regulate gene conversion in an AID independent manner.