Annica Mårtensson

Receptor Editing and Marginal Zone B Cell Development Are Regulated by the Helix-Loop-Helix Protein, E2A

Previous studies have indicated that the E2A gene products are required to initiate B lineage development. Here, we demonstrate that E2A+/− B cells that express an autoreactive B cell receptor fail to mature due in part to an inability to activate secondary immunoglobulin (Ig) light chain gene rearrangement. Both RAG1/2 gene expression and RS deletion are severely defective in E2A+/− mice. Additionally, we demonstrate that E2A+/− mice show an increase in the proportion of marginal zone B cells with a concomitant decrease in the proportion of follicular B cells. In contrast, Id3-deficient splenocytes show a decline in the proportion of marginal zone B cells. Based on these observations, we propose that E-protein activity regulates secondary Ig gene rearrangement at the immature B cell stage and contributes to cell fate determination of marginal zone B cells. Additionally, we propose a model in which E-proteins enforce the developmental checkpoint at the immature B cell stage.

Spi-C, a Novel Ets Protein That Is Temporally Regulated during B Lymphocyte Development

A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 κ promoter κY element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a κY element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.

FGD2, a CDC42-specific Exchange Factor Expressed by Antigen-presenting Cells, Localizes to Early Endosomes and Active Membrane Ruffles

Members of the Fgd (faciogenital dysplasia) gene family encode a group of critical guanine nucleotide exchange factors (GEFs), which, by specifically activating Cdc42, control cytoskeleton-dependent membrane rearrangements. In its first characterization, we find that FGD2 is expressed in antigen-presenting cells, including B lymphocytes, macrophages, and dendritic cells. In the B lymphocyte lineage, FGD2 levels change with developmental stage. In both mature splenic B cells and immature bone marrow B cells, FGD2 expression is suppressed upon activation through the B cell antigen receptor. FGD2 has a complex intracellular localization, with concentrations found in membrane ruffles and early endosomes. Although endosomal localization of FGD2 is dependent on a conserved FYVE domain, its C-terminal pleckstrin homology domain mediates recruitment to membrane ruffles. FGD2 overexpression promotes the activation of Cdc42 and leads to elevated JNK1 activity in a Cdc42- but not Rac1-dependent fashion. These findings are consistent with a role of FGD2 in leukocyte signaling and vesicle trafficking in cells specialized to present antigen in the immune system.

Tolerance-induced receptor selection: scope, sensitivity, locus specificity, and relationship to lymphocyte-positive selection

Summary:  Receptor editing is a mode of immunological tolerance of B lymphocytes that involves antigen‐induced B‐cell receptor signaling and consequent secondary immunoglobulin light chain gene recombination. This ongoing rearrangement often changes B‐cell specificity for antigen, rendering the cell non‐autoreactive and sparing it from deletion. We currently believe that tolerance‐induced editing is limited to early stages in B‐cell development and that it is a major mechanism of tolerance, with a low‐affinity threshold and the potential to take place in virtually every developing B cell. The present review highlights the contributions from our laboratory over several years to elucidate these features.

PEBP2 and c‐myb sites crucial for λ5 core enhancer activity in pre‐B cells

The λ5 gene is expressed exclusively in precursor (pre‐) B cells where its gene product, as part of the pre‐B cell receptor, is crucial for the proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the λ5 gene. Amongst these is an enhancer, Bλ5, located 5′ of the gene. Here we analyze the λ5 enhancer core, bλ5, which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre‐B cells. We identify a DNA element within bλ5, PEBP2λ5, which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre‐B cells. The PEBP2λ5 site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre‐B cells bind to the PEBP2λ5 element. PEBP2 proteins in mature B cells also bind to the PEBP2λ5 element, implying that if PEBP2 proteins are responsible for the stage‐specific expression, they have to be non‐activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c‐myb, binds to a sequence termed mybλ5 located just upstream of the PEBP2λ5 site in the core enhancer.The mybλ5 element is also crucial for enhancer activity, since mutating the myb site reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c‐myb is expressed at high levels in pre‐B cell lines whereas its expression is down‐regulated in more mature B cell lines. Thus, c‐myb may be involved in determining the stage‐specific expression of the λ5 gene.

Identification of a tissue‐ and differentiation stage‐specific enhancer of the VpreB1 gene

The VpreB and λ5 genes encode proteins that associate non‐covalently to form the so‐called surrogate light (SL) chain. The SL chain complexes with the immunoglobulin heavy chain to form the pre‐B cell receptor, which plays a critical role in B cell development. Expression of the murine SL genes is regulated at the level of transcription initiation. Here, we show that a VpreB1 enhancer is located within the 356 bp immediately upstream of the coding sequence. Interestingly, this region exhibits 96 % identity to the upstream region of VpreB2. Deletion mapping located the enhancer to between positions − 214 and − 47 (+ 1 is the 5′‐most transcription initiation site). The enhancer is tissue and differentiation stage specific, and is composed of several DNA elements that are important for its activity. We also show that a transcription factor, early B cell factor, binds to two such elements, and that at least one of these sites is involved in determining enhancer activity.

Haplotype exclusion and receptor editing: irreconcilable differences?

Features of antibody genes and their regulation hinder two properties thought to be critical for clonal selection: haplotype exclusion and receptor diversity. These properties include: (1) the retention of multiple independent L-chain isotypes, which compounds the problem of allelic exclusion with one of isotype exclusion; (2) the process of receptor editing, in which recombination continues in cells already expressing antigen receptors; and (3) non-random associations and quasi-ordered rearrangements of the elements that generate light chain genes, which promote editing at the expense of allelic exclusion and receptor diversification. In contrast, heavy chain gene structure seems to promote haplotype exclusion and receptor diversity. It appears that requirements of receptor selection, such as the need for receptor editing as an immune tolerance mechanism and positive selection as a quality control checkpoint for receptor functionality, impose independent selections that shape the organization and regulation of the antibody genes. Despite these features, B cell development still achieves a significant level of phenotypic haplotype exclusion, suggesting that there is indeed significant selection for antibody monospecificity that is accommodated along with receptor editing. Thus, the immune system achieves both receptor selection and clonal selection, despite their partly antagonistic mechanisms.

PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer’s disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5′ exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.Nemazee and colleagues show that PLD3 and PLD4 are endolysosomal exonucleases that digest ingested nucleic acids and thereby prevent activation of endosomal TLRs. Mice that lack PLD3 and PLD4 develop autoinflammatory disease.