Ligand-independent signaling during early avian B cell development

Surface immunoglobulin (sIg) expression has been conserved as a critical checkpoint in B lymphocyte development. In the chicken embryo, only sIg⁺ B cells are selectively expanded in the bursa of Fabricius, a primary lymphoid organ unique to the avian species. We have previously demonstrated that an interaction between the antigen-binding sites of sIg and a specific bursal ligand(s) is not required to regulate this developmental checkpoint. Rather, the requirement for sIg expression can be attributed to the surface expression of the Igα/β heterodimer associated with sIg. More specifically, ligand-independent signaling downstream of the Igα cytoplasmic domain drives all bursal stages of B cell development during embryogenesis. We discuss here a site-directed mutagenesis approach to identify the critical membrane proximal events involved in ligand-independent signaling during B cell development.     

The complement system in B cell regulation

Early studies of animals bearing natural deficiencies in complement C3 and C4 and mice transiently deficient in C3 suggested that the complement system played a role in humoral immunity. Identification and characterization of the complement receptors CD21 and CD35 and their expression on B lymphocytes provided evidence for a direct role for complement in "linkage of innate and adaptive immunity". More recent studies of mice bearing targeted deficiencies in complement proteins C3, C4 or the receptors CD21/CD35 has confirmed the importance of complement in B cell responses in vivo and extended our understanding to distinct stages in B cell differentiation in which complement participates in humoral immunity. In this review, a role for complement is described in five distinct stages of B cell differentiation.     

Feedback regulation of murine Ly-1 B cell development

Studies presented here, conducted with allotype homozygotes, demonstrate the existence of a feedback mechanism that regulates development of Ly-1 B cells from immature progenitors. In the preceding study (P. A. Lalor et al., Eur. J. Immunol. 1989. 19:501), conducted with allotype heterozygotes, we showed that treating neonates with monoclonal antibody to the paternal allotype IgM depletes roughly half of the neonatal B cell population (i.e. those expressing the paternal IgM allotype) and that paternal allotype Ly-1 B cells specificically remain depleted for the life of the animal. Here we show that treating allotype homozygotes with the same antibody depletes all (rather than half) of the B cells and that, under these conditions, relatively normal numbers of Ly-1 B cells reappear shortly after the treatment antibody disappears. The recovery, we also show, is prevented by restoring allotype-congenic Ly-1 B cells to the treated homozygotes, i.e. by reconstituting treated neonates with allotype-congenic peritoneal cells, sorted Ly-1 B cells or a monoclonal population of Ly-1 B "tumor" cells. These findings in essence reveal a feedback mechanism through which mature Ly-1 B cells prevent further Ly-1 B cell development from Ig- precursors. This feedback regulation is independent of Ig secretion by the mature Ly-1 B cells, since the monoclonal Ly-1 B "tumor" population that prevents endogenous Ly-1 B development does not secrete Ig. Furthermore, it appears to be independent of Ly-1 B surface Ig specificity, since a monoclonal population is sufficient to block all Ly-1 B cell development. This mechanism appears to operate normally to fix the composition of the Ly-1 B population, which survives through self-replenishment in adults, in accord with conditions that influence Ly-1 B development during neonatal life.     

Regulation of immunoglobulin light chain gene rearrangements during early B cell development in the human.

Southern blot analyses of immunoglobulin light chain gene rearrangements in human leukemias and myelomas indicated that lambda loci in kappa-producing cells are largely unrearranged while kappa loci in lambda producers are often rearranged and inactivated by rearrangements of the kappa-deleting element (KDE). For a systematic analysis of the regulation of light chain rearrangements during early B cell development in normal human B cells also considering functionality of the rearrangements, we used FACS-sorted single naive kappa- and lambda-expressing B cells from peripheral blood of healthy humans. V(kappa)J(kappa) and V(lambda)J(lambda) joints and rearrangements involving the KDE were amplified simultaneously from single cells and sequenced. Whereas only 2 - 3 % of kappa-expressing cells carry V(lambda)J(lambda) joints, nearly all lambda-expressing cells have rearranged kappa loci and indeed carry V(kappa)J(kappa) joints. The V(kappa)J(kappa) joints in lambda-expressing cells exhibit preferential J(kappa)4 and J(kappa)5 over J(kappa)1 and J(kappa)2 usage compared to kappa-expressing cells. Thirty percent of the V(kappa)J(kappa) joints in lambda producers are rearranged in-frame. These data indicate extensive sequential V(kappa)-J(kappa) rearrangements and inactivation of functional V(kappa)J(kappa) joints in lambda-expressing cells, presumably before V(lambda)J(lambda) joining.     

2,4-Dinitrophenyl (DNP)-specific continuous B cell lines as a model system for studying B cell activation and tolerance

Various model systems have been used to study isolated B cell response to receptor cross-linking and to lymphokines. Although each model is useful it is advantageous to have continuous cell lines of nonmalignant antigen-specific B lymphocytes to study antigen-induced B cell function. We further studied the characteristics of the 2,4-dinitrophenyl (DNP)-specific continuous B lymphocyte lines which we previously described (J. Exp. Med. 1983. 157:342). If the cell line lymphocytes are cultured with the antigen DNP-Ficoll without the presence of T cell factors or filler cells they do not produce an immune response above background, but the addition of supernatant from EL4 lymphoma and irradiated normal spleen filler cells results in a 7- to 10-fold increase in plaque-forming cells. The kinetics of the immune response is the same as that seen with normal B cells. Each cell line has a majority of cells which are small surface (s)IgM- lymphocytes which have cytoplasmic IgM and react with 14.8 antibody. There are also large sIgM+-bearing cells, which may be either in the resting or activated state. Some of the sIgM+ cells also bear IgD and Ia antigens but they do not bear IgG. From these studies we conclude that the continuously growing antigen-specific B cell lines can be a useful model to study B cell function.     

B cell precursors are present in the thymus during early development

An in vitro system for transforming immature lymphoid cells present in the thymus at early development has been established. By phenotype analysis of the transformants obtained, we observed that B cell precursors, susceptible to Abelson murine leukemia virus (A-MuLV)- or Harvey murine sarcoma virus (H-MuSV)-induced lymphogenesis, were present at high frequency in the fetal thymus of BALB/c mice. These precursors recolonized alymphoid thymus lobes in vitro, as do T cell precursors. It was further observed that B precursors in the fetal liver were also capable of recolonizing alymphoid thymus lobes and were stored in a thymic environment. These results suggest that stroma cells of the fetal thymus may possess the capacity to support the growth of B precursors. On the other hand, B cell precursors sensitive to the viral transformation were undetectable in the fetal thymus of C57BL/6, although immunohistochemical analysis suggested their presence. However, in the fetal liver of the same strain, B precursors recolonizing alymphoid thymus in vitro were sensitive to the viral transformation. Based on these results, we will discuss both the role and fate of thymic B precursors. In addition, we also obtained T cell lymphomas at different stages of differentiation from the fetal thymus of C57BL/6 infected with A-MuLV or H-MuSV. These data indicate the usefulness of our system in establishing cell lines derived from intrathymic lymphogenesis at early development.     

Antibody regulation of B cell development

Antibodies on the surface of B lymphocytes trigger adaptive immune responses and control a series of antigen-independent checkpoints during B cell development. These physiologic processes are regulated by a complex of membrane immunoglobulin and two signal transducing proteins known as Ig alpha and Ig beta. Here we focus on the role of antibodies in governing the maturation of B cells from early antigen-independent through the final antigen-dependent stages.     

Expression and regulation of ROR-1 during early avian limb development

ROR-1 is a member of the ROR family of tyrosine kinase like orphan receptors and is highly conserved among various species. We have isolated the chick ROR-1 (cROR-1) and show that cROR-1 expression is high and restricted to the proximal limb region until HH-stage 25. At later stages, expression spreads towards the distal limb region. In order to determine the signals that control cROR-1 expression, factors known to be involved in limb patterning (FGFs, BMPs, SHH, retinoic acid) were applied to the developing limb. Whereas neither FGFs, BMPs, nor SHH affected cROR-1 expression, upregulation could be achieved by ectopic application of retinoic acid to the distal limb region. As retinoic acid also upregulated retinoic acid receptor beta (Rar-), we assume that cROR-1 upregulation is mediated by Rar-. We conclude that ROR-1 signaling is an independently regulated pathway, which is involved in late rather than early limb development.     

Expression and regulation of HTRA1 during chick and early mouse development

HTRA1, a member of the high temperature requirement factor A family, is a secreted serine protease that can bind to and inactivate members of the transforming growth factor-beta (TGFbeta) family, modulate insulin-like growth factor signaling and stimulate long range fibroblast growth factor (FGF) signaling in Xenopus. In vertebrates, so far homologues from mouse, human, and Xenopus have been cloned and studied. Here we report the cloning of the chicken HTRA1 homologue from a screen for FGF8 inducible genes in chick facial mesenchyme. We characterize its expression pattern from gastrulation (Hamburger and Hamilton stage 4) to day 4 of development, and in forming inner organs and limbs. We show that chick HTRA1 has a dynamic expression pattern that differs significantly from the expression of its mouse homolog. We, furthermore, demonstrate that FGF signaling is necessary and sufficient for HTRA1 expression in chick facial and forelimb mesenchyme, but is not required for HTRA1 expression in HH11 embryos.     

Immunoglobulin kappa variable region gene selection during early human B cell development in health and systemic lupus erythematosus

The unique specificity of the B cell receptor is generated by an ordered sequence of gene rearrangement events. Once IGH genes have rearranged, rearrangement at the IGK locus is initiated followed by the IGL locus if functional IGK rearrangement is not achieved. Receptor specificity can subsequently be altered by secondary light chain editing based on the features of the heavy and light chain combination. The final profile of expressed genes is not random and biases in this profile are associated with several autoimmune diseases. However, how and when biases are created is not known.To increase our understanding of the processes of selection and editing of IGK rearrangements, we compared four groups of rearrangements of IGK acquired by next generation sequencing. First, expressed rearrangements of IGK from cDNA of IGK expressing B cells. Second, productive rearrangements of IGK from DNA of the same kappa expressing B cells. Third, non-productive rearrangements of IGK from DNA of IGK and IGL expressing B cells, and fourth productively rearranged IGK from DNA of IGL expressing B cells. The latter group would have been rejected during B cell development in favour of rearrangement at the IGL locus and are therefore selected against.We saw evidence that rearranged IGK segments can be selected at a checkpoint where the decision to rearrange the IGL locus is made. In addition, our data suggest that mechanisms regulating the expression or not of IGK rearrangements may also contribute to repertoire development and also that this latter component of the selection process is defective in SLE.     

Single-molecule methods for studying gene regulation in vivo

The recent emergence of new experimental tools employing sensitive fluorescence detection in vivo has made it possible to visualize various aspects of gene regulation at the single-molecule level in the native, intracellular context. In this review, we will first describe general considerations for in vivo, single-molecule fluorescence detection of DNA, mRNA, and protein molecules involved in gene regulation. We will then give an overview of the rapidly evolving suite of molecular tools available for observing gene regulation in vivo and discuss new insights they have brought into gene regulation.     

Btg1 and Btg2 gene expression during early chick development

Btg/Tob genes encode for a new family of proteins with antiproliferative functions, which are also able to stimulate cell differentiation. Btg1 and Btg2 are the most closely related members in terms of gene sequence. We analyzed their expression patterns in avian embryos by in situ hybridization, from embryonic day 1 to 3. Btg1 was distinctively expressed in the Hensen's node, the notochord, the cardiogenic mesoderm, the lens vesicle, and in the apical ectodermal ridge and mesenchyme of the limb buds. On the other hand, Btg2 expression domains included the neural plate border, presomitic mesoderm, trigeminal placode, and mesonephros. Both genes were commonly expressed in the myotome, epibranchial placodes, and dorsal neural tube. The results suggest that Btg1 and Btg2 are involved in multiple developmental processes. Overlapping expression of Btg1 and Btg2 may imply redundant functions, but unique expression patterns suggest also differential regulation and function.     

The pre-B cell receptor and its function during B cell development

The process of B cell development in the bone marrow occurs by the stepwise rearrangements of the V, D, and J segments of the Ig H and L chain gene loci. During early B cell genesis, productive Ig H chain gene rearrangement leads to assembly of the pre-B cell receptor (pre-BCR), which acts as an important checkpoint at the pro-B/preB transitional stage. The pre-BCR, transiently expressed by developing precursor B cells, comprises the Ig muH chain, surrogate light (SL) chains VpreB and lambda5, as well as the signal-transducing heterodimer Ig alpha/Ig beta. Signaling through the pre-BCR regulates allelic exclusion at the Ig H locus, stimulates cell proliferation, and induces differentiation to small post-mitotic pre-B cells that further undergo the rearrangement of the Ig L chain genes. Recent advances in elucidating the key roles of pre-BCR in B cell development have provided a better understanding of normal B lymphopoiesis and its dysregulated state leading to B cell neoplasia.     

Differential regulation of mouse B cell development by transforming growth factor beta1

Transforming growth factor beta (TGFbeta) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFbeta1-/- mice. To evaluate TGFbeta responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7) +/- TGFbeta. Picomolar doses of TGFbeta1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFbeta1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFbeta1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.