Elias Hobeika

Natural Aryl Hydrocarbon Receptor Ligands Control Organogenesis of Intestinal Lymphoid Follicles

Nutrients can shape immune system development function through aryl hydrocarbon receptor signaling. Innate lymphoid cells (ILC) expressing the transcription factor RORγt induce the postnatal formation of intestinal lymphoid follicles and regulate intestinal homeostasis. RORγt+ ILC express the aryl hydrocarbon receptor (AhR), a highly conserved, ligand-inducible transcription factor believed to control adaptation of multicellular organisms to environmental challenges. We show that AhR is required for the postnatal expansion of intestinal RORγt+ ILC and the formation of intestinal lymphoid follicles. AhR activity within RORγt+ ILC could be induced by dietary ligands such as those contained in vegetables of the family Brassicaceae. AhR-deficient mice were highly susceptible to infection with Citrobacter rodentium, a mouse model for attaching and effacing infections. Our results establish a molecular link between nutrients and the formation of immune system components required to maintain intestinal homeostasis and resistance to infections.

Testing gene function early in the B cell lineage in mb1-cre mice

The mb1 gene encodes the Ig-α signaling subunit of the B cell antigen receptor and is expressed exclusively in B cells beginning at the very early pro-B cell stage in the bone marrow. We examine here the efficacy of the mb1 gene as a host locus for cre recombinase expression in B cells. We show that by integrating a humanized cre recombinase into the mb1 locus we obtain extraordinarily efficient recombination of loxP sites in the B cell lineage. The results from a variety of reporter genes including the splicing factor SRp20 and the DNA methylase Dnmt1 suggest that mb1-cre is probably the best model so far described for pan-B cell-specific cre expression. The availability of a mouse line with efficient cre-mediated recombination at an early developmental stage in the B lineage provides an opportunity to study the role of various genes specifically in B cell development and function.

B Cell Progenitors Are Arrested in Maturation but Have Intact VDJ Recombination in the Absence of Ig-α and Ig-β

Ig-α and Ig-β mediate surface expression and signaling of diverse B cell receptor complexes on precursor, immature, and mature B cells. Their expression begins before that of the Ig chains in early progenitor B cells. In this study, we describe the generation of Ig-α-deficient mice and their comparative analysis to mice deficient for Ig-β, the membrane-IgM, and recombination-activating gene 2 to determine the requirement of Ig-α and Ig-β in survival and differentiation of pro-B cells. We find that in the absence of Ig-α, B cell development does not progress beyond the progenitor stage, similar to what is observed in humans lacking this molecule. However, neither in Ig-α- nor in Ig-β-deficient mice are pro-B cells impaired in V(D)J recombination, in the expression of intracellular Ig μ-chains, or in surviving in the bone marrow microenvironment. Finally, Ig-α and Ig-β are not redundant in their putative function, as pro-B cells from Ig-α and Ig-β double-deficient mice are similar to those from single-deficient animals in every aspect analyzed.

Autoreactive B Cell Receptors Mimic Autonomous Pre-B Cell Receptor Signaling and Induce Proliferation of Early B Cells

The majority of early immature B cells express autoreactive B cell receptors (BCRs) that are, according to the current view, negatively selected to avoid the production of self-reactive antibodies. Here, we show that polyreactive BCRs, which recognize multiple self-antigens, induced autonomous signaling and selective expansion of B cell precursors in a manner comparable to the pre-BCR. We found that the pre-BCR was capable of recognizing multiple self-antigens and that a signaling-deficient pre-BCR lacking the non-Ig region of the surrogate-light-chain component lambda5 was rescued by the complementarity-determining region 3 derived from heavy chains of polyreactive receptors. Importantly, bone marrow B cells from mice carrying Ig transgenes for an autoreactive BCR showed increased cell-cycle activity, which could not be detected in cells lacking the transgenic BCR. Together, the pre-BCR has evolved to ensure self-recognition because autoreactivity is required for positive selection of B cell precursors.

The kinase Syk as an adaptor controlling sustained calcium signalling and B‐cell development

Upon B‐cell antigen receptor (BCR) activation, the protein tyrosine kinase Syk phosphorylates the adaptor protein SH2 domain‐containing leukocyte protein of 65 kDa (SLP‐65), thus coupling the BCR to diverse signalling pathways. Here, we report that SLP‐65 is not only a downstream target and substrate of Syk but also a direct binding‐partner and activator of this kinase. This positive feedback is mediated by the binding of the SH2 domain of SLP‐65 to an autophosphorylated tyrosine of Syk. The mutant B cells that cannot form the Syk/SLP‐65 complex are defective in BCR‐induced extracellular signal‐regulated kinase, nuclear factor κ B and nuclear factor of activated T cells, but not Akt activation, and are blocked in B‐cell development. Furthermore, we show that formation of the Syk/SLP‐65 complex is required for sustained Ca2+ responses in activated B cells. We suggest that after activation and internalization of the BCR, Syk remains active as part of a membrane‐bound Syk/SLP‐65 complex controlling sustained signalling and calcium influx.

Role of PI3K in the generation and survival of B cells

Summary:  Engagement of the B‐cell antigen receptor (BCR) or its precursor, the pre‐BCR, induces a cascade of biochemical reactions that regulate the differentiation, selection, survival, and activation of B cells. This cascade is initiated by receptor‐associated tyrosine kinases that activate multiple downstream signaling pathways. Since it is required for metabolism, cell growth, development, and survival, the activation of phosphoinositide 3‐kinase (PI3K)‐dependent pathways represents a crucial event of BCR/pre‐BCR signaling. The phosphorylated substrates of the PI3K promote specific recruitment of selected signaling proteins to the plasma membrane, where important signaling complexes are formed to mediate the above‐mentioned biological processes. Here, we review the principles of PI3K signaling and highlight the role of an important PI3K‐driven module in VDJ recombination of immunoglobulin (Ig) genes during early B‐cell development as compared with class switch recombination of Ig genes in mature B cells after activation by specific antigens. Furthermore, we discuss the role of PI3K in the survival of mature B cells, which is strictly dependent on BCR expression and basal BCR signaling.

Induction of B-cell development in adult mice reveals the ability of bone marrow to produce B-1a cells

To study B-cell development from bone marrow (BM), we generated recombination-activating gene 1 (Rag1)-targeted mice lacking mature lymphocytes. B-cell development can be induced in such mice by B cell-specific restoration of a functional Rag1 transcription unit. Follicular and marginal zone B cells populated the spleen when Rag1 expression was permitted. Notably, the peritoneal cavity was dominated by bona fide B-1a cells, as judged by surface markers and functional properties. These BM-derived B-1a cells exhibited a polyclonal VDJ repertoire with substantial N nucleotide insertions. Nevertheless, physiologic frequencies of phosphatidylcholine-specific B cells were detected. Importantly, the BM of young and 5-month-old mice was indistinguishable with regard to the potential to generate B-1a cells.

B cell homeostasis and plasma cell homing controlled by Krüppel-like factor 2

Krüppel-like factor 2 (KLF2) controls T lymphocyte egress from lymphoid organs by regulating sphingosin-1 phosphate receptor 1 (S1Pr1). Here we show that this is not the case for B cells. Instead, KLF2 controls homeostasis of B cells in peripheral lymphatic organs and homing of plasma cells to the bone marrow, presumably by controlling the expression of β7-integrin. In mice with a B cell-specific deletion of KLF2, S1Pr1 expression on B cells was only slightly affected. Accordingly, all splenic B cell subsets including B1 cells were present, but their numbers were increased with a clear bias for marginal zone (MZ) B cells. In contrast, fewer peyers patches harboring fewer B cells were found, and fewer B1 cells in the peritoneal cavity as well as recirculating B cells in the bone marrow were detected. Upon thymus-dependent immunization, IgG titers were diminished, and antigen-specific plasma cells were absent in the bone marrow, although numbers of antigen-specific splenic plasmablasts were normal. KLF2 plays also a role in determining the identity of follicular B cells, as KLF2-deficient follicular B cells showed calcium responses similar to those of MZ B cells and failed to down-regulate MZ B cell signature genes, such as CD21 and CXCR7.

B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk

Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation. How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy. Using a high-resolution proximity ligation assay (PLA) to monitor the conformation of the BCR and its interactions with co-receptors at a 10–20 nm resolution, we provide direct evidence for the opening of BCR dimers during B cell activation. We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling. Furthermore, we found that on resting B cells, the coreceptor CD19 is in close proximity with the IgD-BCR and on activated B cells with the IgM-BCR, indicating nanoscale reorganization of receptor clusters during B cell activation. DOI: http://dx.doi.org/10.7554/eLife.02069.001

Identification of a Pre-BCR Lacking Surrogate Light Chain

SLP-65−/− pre-B cells show a high proliferation rate in vitro. We have shown previously that λ5 expression and consequently a conventional pre-B cell receptor (pre-BCR) are essential for this proliferation. Here, we show that pre-B cells express a novel receptor complex that contains a μ heavy chain (μHC) but lacks any surrogate (SL) or conventional light chain (LC). This SL-deficient pre-BCR (SL−pre-BCR) requires Ig-α for expression on the cell surface. Anti-μ treatment of pre-B cells expressing the SL−pre-BCR induces tyrosine phosphorylation of substrate proteins and a strong calcium (Ca2+) release. Further, the expression of the SL−pre-BCR is associated with a high differentiation rate toward κLC-positive cells. Given that B cell development is only partially blocked and allelic exclusion is unaffected in SL-deficient mice, we propose that the SL−pre-BCR is involved in these processes and therefore shares important functions with the conventional pre-BCR.