Olli Lassila

The origin of lymphoid stem cells studied in chick yolk sac–embryo chimaeras

THE development of lymphoid organs in the chick embryo is dependent on colonisation by extrinsic stem cells1–6, some of which are present in the yolk sac blood islands, as Moore and Owen demonstrated by transplanting 7-d yolk sac cells into 14-d-old irradiated chicken embryos2. These authors demonstrated population of lymphoid organs by donor-derived cells, and suggested that all haemopoietic stem cells are formed in the early yolk sac3. This interpretation has been challenged7–11, particularly on the basis of results obtained in quail–chick chimaeric germs7. We present here further evidence, based on the use of chick–chick yolk sac–embryo chimaeras, that lymphoid stem cells in the chicken do not originate primarily in the yolk sac.

Bacterial 16S rDNA polymerase chain reaction in the detection of intra-amniotic infection

Objective Bacterial polymerase chain reaction (PCR) was used to detect early subclinical intra‐amniotic infection. We used universal primers which amplify a DNA fragment of 16S ribosomal DNA (rDNA) from all known bacteria and sequenced the positive samples to identify the bacterial species.

Development of Mitogen Responding T Cells and Natural Killer Cells in the Human Fetus

Human T lymphocytes acquire immunocompetence during fetal life, even though at the time of full-term birth their functional capacity may not be equal to that ofadult lymphocytes (Toivanen et al. 1978, Stiehm et al. 1979, Stites & Pavia 1979, Toivanen & Granberg 1980). In this paper we will summarize and discuss recent studies carried out in our laboratory on the fetal development of human T cells responding to mitogens and of cytotoxic cells, paying particular attention to the appearance of natural killer (NK) cell activity.

Chicken B-Cell-Activating Factor: Regulator of B-Cell Survival in the Bursa of Fabricius

Previous studies on mammals have demonstrated that a tumour necrosis factor family member, B‐cell‐activating factor (BAFF) (BlyS, TALL‐1), is mainly produced by myeloid and dendritic cells and that BAFF promotes B‐cell differentiation and survival in a paracrine fashion. We have recently shown that BAFF is upregulated at the bursal stage of the avian B‐cell development. We now show that the avian bursal B cells and B‐cell lines, RP‐9, RP‐13 and DT40, express chicken BAFF (cBAFF). In situ hybridization confirms strong cBAFF expression within the bursal follicles. Like mammals, cBAFF is expressed in the avian myeloblast and myelomonocytic cell lines but not in the peripheral blood αβ and γδ T cells. The binding of recombinant human BAFF (hBAFF) to the bursal B‐cells indicates a conserved receptor–ligand binding. Furthermore, the recombinant hBAFF has a positive effect on bursal cell proliferation and transiently inhibits cell death in vitro. In conclusion, cBAFF is highly conserved structurally, but as a novel observation we suggest cBAFF to function in an autocrine fashion to promote the growth and maturation of follicular B cells in bursa of Fabricius.

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.