Kalle-Pekka Nera

Alternate pathways for Bcl6-mediated regulation of B cell to plasma cell differentiation

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.

Aiolos Controls Gene Conversion and Cell Death in DT40 B Cells

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.

Pax5 – a Critical Inhibitor of Plasma Cell Fate

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.

Ikaros has a crucial role in regulation of B cell receptor signaling

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 Cγ2 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.

Concerted action of Helios and Ikaros controls the expression of the inositol 5-phosphatase SHIP

Ikaros family transcription factors have a key role in lymphoid development, and their aberrant function contributes to a multitude of lymphoid malignancies. Ikaros and Helios bind to similar DNA sequences, and Helios associates with Ikaros‐containing chromatin remodeling complexes. Previously, we have shown that loss of Ikaros leads to diminished BCR‐signaling strength. In this study, we describe a Helios‐deficient chicken DT40 B‐cell line with a BCR signaling phenotype that is the opposite to that of Ikaros‐deficient cells. In contrast to Ikaros‐deficient cells, Helios−/− B cells exhibit increased calcium release to the cytoplasm after BCR crosslinking, but diminished BCR‐induced phosphorylation of signaling molecules. The inositol 5‐phosphatase SHIP, an important regulator in several signaling pathways, is differentially expressed in Ikaros‐ and Helios‐deficient cells. In the absence of Ikaros, SHIP is upregulated, whereas Helios deficiency leads to the downregulation of SHIP expression. We also show with ChIP that Ikaros binds to the promoter of the INPP5D gene‐encoding SHIP. Considering the critical role of SHIP in the BCR signaling pathway, our findings provide insight into the mechanism of how both Helios and Ikaros are involved in the regulation of BCR signaling.

Both normal and leukemic B lymphocytes express multiple isoforms of the human Aiolos gene

Aiolos is a chromatin remodeling transcription regulator that plays an antiproliferative role in B lymphocyte function. In contrast to the related Ikaros factors, mammalian Aiolos has not beenreported to generate splice variants. In addition, although human leukemic lymphoblasts express non‐DNA‐binding Ikaros isoforms with potential dominant negative effect on other interacting factors,the role of Aiolos in human lymphoid disorders has remained obscure. To address the question, why Aiolos should delineate from Ikaros in such a marked way, we have here analyzed whether also human Aiolos could generate alternate isoforms. According to the results obtained, both normal and neoplastic B lineage cells were found to express at least five novel Aiolos variants. Also structurally dominant negative variants with less than three DNA‐binding domains were identified. In conclusion, given the multiplicity of also human Aiolos isoforms and thereby the evidently more intricate contribution of Aiolos to the chromatin remodeling machinery, it is suggested, that not only Ikaros, but also Aiolos could participate in a more versatile manner in the regulation of B lymphocyte function.

DT40 mutants: a model to study transcriptional regulation of B cell development and function

A key issue in understanding the hematopoietic system and B cell biology is to define the function of transcription factors. B lymphocyte development and function is controlled by a hierarchy of transcription factors including PU.1, Ikaros, E2A, EBF, Pax5 and Aiolos. Mouse knockout models provide information about the developmental and physiological importance of the disrupted gene. However, an early block in the development or a lethal phenotype prevents the studies of the functional importance of the gene at the later developing system such as the immune system. The chicken B cell line DT40 is used to circumvent these problems. Studies with DT40 have revealed a role for Ikaros transcription factor in B cell receptor signaling and Aiolos has been shown to regulate immunoglobulin gene conversion and cell survival. On the other hand, findings with Pax5 deficient mutants support DT40 targeting system as a valid model for the plasma cell differentiation and demonstrate the genetic plasticity of the cell line. This system is an excellent model to study transcription factors in B cell specific functions, antibody production and B cell differentiation.

Avian Helios and Evolution of the Ikaros Family

Helios (Znfn1a2) is an Ikaros‐related lymphoid regulatory protein with possible involvement in T‐cell development and function as well as in the early events of haematopoietic stem cell differentiation. To evaluate the role of Helios in avian haemato/lymphopoiesis, we have characterized the avian Helios gene. In contrast to studies in mouse and human, we have found that the highly conserved avian Helios encodes a novel exon and three isoforms. Furthermore, the avian Helios expression precedes Ikaros in the ontogeny, being present already on the first day of embryonic development. Additionally, expression in the bursa of Fabricius, germinal centres and B‐cell lines suggests a role for Helios also in the B‐cell lineage. Phylogenetic studies of the Ikaros family along with data on paralogous chromosome segments in the human genome connect the expansion of the Ikaros family and thus possibly the emergence of the adaptive immune system with the putative second round of genome duplications and indicate that the Ikaros gene family is linked with the Hox gene clusters.

Regulation of B Cell to Plasma Cell Transition within the Follicular B Cell Response

Persistent humoral immunity depends on the follicular B cell response and on the generation of somatically mutated high‐affinity plasma cells and memory B cells. Upon activation by an antigen, cognately activated follicular B cells and follicular T helper (TFH) cells initiate germinal centre (GC) reaction during which high‐affinity effector cells are generated. The differentiation of activated follicular B cells into plasma cells and memory B cells is guided by complex selection events, both at the cellular and molecular level. The transition of B cell into a plasma cell during the GC response involves alterations in the microenvironment and developmental state of the cell, which are guided by cell‐extrinsic signals. The developmental cell fate decisions in response to these signals are coordinated by cell‐intrinsic gene regulatory network functioning at epigenetic, transcriptional and post‐transcriptional levels.

The Ikaros Family and the Development of Early Intraembryonic Hematopoietic Stem Cells

It is currently well established that across evolutionarily distant species the first blood-forming stem cells appear in the blood islands of both the extraembryonic yolk sac and the embryo proper. Following this initial and only transient generation of primitive erythroid-lineage cells, the major hematopoietic regions are thereafter located in the intraembryonic mesenchyme of the early embryos. Taking advantage of avian yolk sac-embryo chimeras it was originally shown that lymphoid cells are of intraembryonic origin [1, 2]. Two decades after the avian grafting experiments, the murine para-aortic splanchnopleura (P-Sp) and the aorta-gonad-mesonephrosregion (AGM) were functionally characterized to have lymphoid potential [3, 4]. These results confirmed the intraembryonic sources for murine lymphopoiesis. Thus, mesodermal foci, i.e. the splanchnopleura and the ventrally derived intraaortic clusters as well as the later appearing dorsal mesentery-derived para-aortic loci, initiate development of hematopoietic stem cells (HSCs). Although the AGM region is already known to harbor committed progenitors, and lymphoid potential has been shown to reside even in the para-aortic splanchnopleura, the exact time schedule of fate decisions is still rather controversial and unresolved. Contradictory evidence exists concerning the role of the P-Sp/AGM area on one hand as a de novo generator of intraembryonic hematopoietic stem cells and on the other hand as a site of hematopoietic differentiation. It has been postulated that these early hematopoietic environments only give rise to hematopoietic stem cells and further differentiation occurs in the lymphoid organs after colonization [5]. By contrast however, there are data showing the presence of committed lymphocyte progenitors not only in the fetal liver, but also in the murine AGM [6]. These results that are based on a clonal assay system support the idea of early lineage-determination in the lymphoid system. Taken together, these findings confirm not only the intraembryonic origin of the lymphoid cells, but also support the idea of early fate determination occurring before seeding of the primordial lymphoid organs.