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Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed intracellular prolyl peptidase implicated in immunoregulation. However, its physiological relevance in the immune system remains largely unknown. We investigated the role of DPP9 enzyme in immune system by characterizing DPP9 knock-in mice expressing a catalytically inactive S729A mutant of DPP9 enzyme (DPP9 ki/ki mice). DPP9 ki/ki mice show reduced number of lymphoid and myeloid cells in fetal liver and postnatal blood but their hematopoietic cells are fully functional and able to reconstitute lymphoid and myeloid lineages even in competitive mixed chimeras. These studies demonstrate that inactivation of DPP9 enzymatic activity does not lead to any perturbations in mouse hematopoiesis., (Copyright © 2018 European Federation of Immunological Societies. All rights reserved.)

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.

B cell activating factor (BAFF) provides B cells with essential survival signals. It binds to three receptors: BAFFR, TACI, and BCMA that are differentially expressed by B cell subsets. BAFFR is early expressed in circulating B cells and provides key signals for further maturation. Here, we report that highly regulated BAFFR processing events modulate BAFF responses. BAFFR processing is triggered by BAFF binding in B cells co-expressing TACI and it is executed by the metalloproteases ADAM10 and ADAM17. The degree of BAFF oligomerization, the expression of ADAM proteins in different B cell subsets, and the activation status of the cell determine the proteases involved in BAFFR processing. Inhibition of ADAM10 augments BAFF-dependent survival of primary human B cells, whereas inhibition of ADAM17 increases BAFFR expression levels on germinal center B cells. Therefore, BAFF-induced processing of BAFFR regulates BAFF-mediated B cell responses in a TACI-dependent manner., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Up to now long-term in vitro growth of pro-B cells was thought to require stromal cells. However, here we show that fetal liver (FL) and bone marrow (BM) derived pro-B cells can be propagated long-term in stromal cell-free cultures supplemented with IL-7, stem cell factor and FLT3 ligand. Within a week, most cells expressed surface CD19, CD79A, λ5, and VpreB antigens and had rearranged immunoglobulin D-J heavy chain genes. Both FL and BM pro-B cells reconstituted the B-cell compartments of immuno-incompetent Rag2-deficient mice, with FL pro-B cells generating follicular, marginal zone (MZB) and B1a B cells, and BM pro-B cells giving rise mainly to MZB cells. Reconstituted Rag2-deficient mice generated significant levels of IgM and IgG antibodies to a type II T-independent antigen; mice reconstituted with FL pro-B cells generated surprisingly high IgG 1 titers. Finally, we show for the first time that mice reconstituted with mixtures of pro-B and pro-T cells propagated in stromal cell-free in vitro cultures mounted a T-cell-dependent antibody response. This novel stromal cell-free culture system facilitates our understanding of B-cell development and might be applied clinically., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Flt3 ligand (Flt3L) promotes survival of lymphoid progenitors in the bone marrow and differentiation of dendritic cells (DCs), but its role in regulating innate lymphoid cells (ILCs) during fetal and adult life is not understood. By using Flt3L knockout and transgenic mice, we demonstrate that Flt3L controls ILC numbers by regulating the pool of α4β7(-) and α4β7(+) lymphoid tissue inducer cell progenitors in the fetal liver and common lymphoid progenitors in the bone marrow. Deletion of flt3l severely reduced the number of fetal liver progenitors and lymphoid tissue inducer cells in the neonatal intestine, resulting in impaired development of Peyer's patches. In the adult intestine, NK cells and group 2 and 3 ILCs were severely reduced. This effect occurred independently of DCs as ILC numbers were normal in mice in which DCs were constitutively deleted. Finally, we could show that administration of Flt3L increased the number of NKp46(-) group 3 ILCs in wild-type and even in Il7(-/-) mice, which generally have reduced numbers of ILCs. Taken together, Flt3L significantly contributes to ILC and Peyer's patches development by targeting lymphoid progenitor cells during fetal and adult life., (Copyright © 2016 by The American Association of Immunologists, Inc.)

A widely accepted model posits that activation of enhancers during differentiation goes through a priming step prior to lineage commitment. To investigate the chronology of enhancer repertoire establishment during hematopoiesis, we monitored epigenome dynamics during three developmental stages representing hematopoietic stem cells, B-cell progenitors and mature B-cells. We find that only a minority of enhancers primed in stem cells or progenitors become active at later stages. Furthermore, most enhancers active in differentiated cells were not primed in earlier stages. Thus, the enhancer repertoire is reshaped dynamically during B-cell differentiation and enhancer priming in early stages does not appear to be an obligate step for enhancer activation. Furthermore, our data reveal that heterochromatin and Polycomb-mediated silencing have only a minor contribution in shaping enhancer repertoires during cell differentiation. Together, our data revisit the prevalent model about epigenetic reprogramming during hematopoiesis and give insights into the formation of gene regulatory networks.

Immunoglobulin genes V(D)J rearrangement during early lymphopoiesis is a critical process involving sequential recombination of the heavy and light chain loci. A number of transcription factors act together with temporally activated recombinases and chromatin accessibility changes to regulate this complex process. Here, we deleted the de novo DNA methyltransferases Dnmt3a and Dnmt3b in early B cells of conditionally targeted mice, and monitored the process of V(D)J recombination. Dnmt3a and Dnmt3b deletion resulted in precocious recombination of the immunoglobulin κ light chain without impairing the differentiation of mature B cells or overall B-cell development. Ex vivo culture of IL-7 restricted early B-cell progenitors lacking Dnmt3a and Dnmt3b showed precocious Vκ-Jκ rearrangements that are limited to the proximal Vκ genes. Furthermore, B-cell progenitors deficient in Dnmt3a and Dnmt3b showed elevated levels of germline transcripts at the proximal Vκ genes, alterations in methylation patterns at Igκ enhancer sites and increased expression of the transcription factor E2A. Our data suggest that Dnmt3a and Dnmt3b are critical to regulate the onset of Igκ light chain rearrangement during early B-cell development., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Protein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations., Methods: Transgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes. Only the soluble form of human IgH-γ1 was used to avoid expression of the membrane Ig-H chain and concomitant allelic exclusion of endogenous murine Ig genes. IgG1 aggregates were generated by different stress conditions such as process-related, low pH and exposure to artificial light., Results: The expression of human Ig proteins induced immunological tolerance to a broad range of human IgG1 molecules in the tg mice. Immunization with IgG1 aggregates demonstrated that soluble oligomers induced by significant light-exposure and carrying neo-epitopes induced a strong immune response in tg mice. In contrast, Ab aggregates alone and monomers with neo-epitopes were not immunogenic., Conclusion: This mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

While in patients with rheumatoid arthritis B-cell repopulation starts within 9 months after rituximab (RTX) therapy, a delayed B-cell repopulation was reported in some RTX-treated patients with ANCA-associated vasculitides (AAV). To date, the frequency of AAV patients with impaired peripheral B-cell regeneration and the mechanisms leading to the constricted regenerative capacity are unknown. We analyzed the B-cell repopulation kinetic in 37 AAV patients treated with RTX followed by maintenance immunosuppressants. We report on serum concentrations of the B-cell-activating factor BAFF, immunoglobulins and B-cell subpopulations in patients that relapsed after RTX. B-cells were re-detectable in only one patient within 9 months after RTX. In 14 patients (41%), B-cell repopulation started later, after a mean observation time of 21 months. Only seven of these patients had detectable B-cells within the first year after RTX. Twenty patients (59%) had no B-cell reconstitution within the observation period. BAFF was increased in RTX-treated AAV patients compared to healthy controls and correlated inversely with peripheral B-cell numbers, IgG- and IgA concentrations. Immunoglobulin concentrations declined significantly after RTX and the IgG concentration correlated with B-cell numbers. Thirteen patients relapsed after RTX. Relapses occurred exclusively either after B-cell reconstitution had started or were accompanied by rising ANCA titres. In relapsed patients, the B-lymphocyte compartment consisted mainly of switched memory B-cells. Our data indicate that RTX treatment can induce secondary immunodeficiency in AAV, with hypogammaglobulinemia and long-lasting B-lymphopenia. Further studies are needed to define the pathophysiology of the impaired B-cell development in RTX-treated AAV patients.

CD19 plays a crucial role in mature B cell development as best exemplified by the finding that CD19 deficient mice have severely reduced mature B cell compartments (Engel et al., 1995; Rickert et al., 1995). In the present study we show that the transition into the mature B cell compartments is heavily dependent on the correct amount of CD19 expression. Thus, Nup-98-HoxB4 immortalized hematopoietic stem cells (HSCs) over-expressing CD19 show upon transplantation an impaired pro/pre B to immature B cell transition in the bone marrow, whereas Nup-98-HoxB4 HSCs expressing a shRNA that down-modulates CD19 expression show upon transplantation a strongly reduced mature B cell compartment. Overall our findings indicate that too high CD19 expression might result into too strong BCR signaling in the bone marrow and therefore causing negative selection. Too low CD19 expression might result into too little BCR signaling and thereby preventing the B cells to enter the mature pool (absence of positive selection)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner.

Aims: Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE(-/-) mouse., Methods and Results: To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE(-/-) mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93(-) CD19(+) B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19(+) B cells, CD4(+) and CD8(+) T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE(-/-) mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93(-) CD19(+) B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment., Conclusion: Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE(-/-) mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.

In mice, interleukin 7, although initially thought to be predominantly a cytokine acting on B cells, appears to have potent survival and growth activity during both B and T lymphopoiesis. Although acting on both lineages at the very immature stages, T cell differentiation becomes independent of IL-7 at the intermediate stages before regaining dependence on IL-7 for survival and proliferation at the mature T cell stage. In contrast, although essential for B lymphopoiesis, mature B cell survival is independent of IL-7. In this review, we focus on and discuss the similarities and differences between the role of IL-7 in these two processes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The TNF family member protein BAFF/BLyS is essential for B cell survival and plays an important role in regulating class switch recombination as well as in the selection of autoreactive B cells. In humans, increased concentrations of soluble BAFF are found in different pathological conditions, which may be as diverse as autoimmune diseases, B cell malignancies, and primary Ab deficiencies (PAD). Because the mechanisms that regulate BAFF levels are not well understood, we newly developed a set of mAbs against human BAFF to study the parameters that determine the concentrations of soluble BAFF in circulation. Patients with PAD, including severe functional B cell defects such as BTK, BAFF-R, or TACI deficiency, were found to have higher BAFF levels than asplenic individuals, patients after anti-CD20 B cell depletion, chronic lymphocytic leukemia patients, or healthy donors. In a comparable manner, mice constitutively expressing human BAFF were found to have higher concentrations of BAFF in the absence than in the presence of B cells. Therefore, our data strongly suggest that BAFF steady-state concentrations mainly depend on the number of B cells as well as on the expression of BAFF-binding receptors. Because most patients with PAD have high levels of circulating BAFF, the increase in BAFF concentrations cannot compensate defects in B cell development and function.

The interaction between BAFF and BAFF-R is crucial for the development of mature B cells. Here, we report that the expression of BAFF-R is first detectable on a fraction of mouse CD19(+) CD93(+) IgM(+) CD23(-) and human CD19(+) CD10(+) IgM(+) BM B cells. This BAFF-R(+) BM B-cell population shows higher levels of surface IgM expression and decreased RAG-2 transcripts than BAFF-R(-) immature B cells. When cultured, mouse BAFF-R(-), but not BAFF-R(+) immature B cells spontaneously undergo B-cell receptor editing. However, BAFF-R(+) immature B cells cultured in the presence of an anti-κ light chain antibody are induced to undergo receptor editing. This receptor editing correlates with down-modulation of surface BAFF-R expression and the up-regulation of RAG-2 at the RNA level. B-cell receptor (BCR) cross-linking on splenic T1 B cells results in down-modulation of the BAFF-R, and receptor editing and RAG-2 up-regulation in a minor fraction of B cells. BCR cross-linking on splenic T2/3 B cells results in partly down and partly up-modulation of BAFF-R expression and no evidence for receptor editing. Overall, our data indicate that BAFF-R expression is tightly regulated during B-cell development in mouse and human and its expression is correlated with positive selection., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The requirement for Notch signaling during T-cell development has been extensively studied. Nevertheless, the developmental stage at which it is required and whether additional signaling pathways are needed are still poorly understood. By using a stromal-cell-free culture system, we show that sorted double-negative 3 (DN3) thymocytes only require a Delta-like-4-induced Notch signal to differentiate into double-positive (DP) cells. This differentiation process is preTCR-α dependent. DN3 cells undergo 4-5 proliferation cycles, and the addition of the chemokine CXCL12 improves proliferation. IL-7 blocks the differentiation of DN3 cells to DP cells but not the Notch-induced proliferation of cultured DN3 cells. The impaired differentiation correlates with an inhibition of Rag-2 up-regulation. Overall, the in vitro stromal-cell-free culture system presented here also provides a powerful and unique tool for studying the mechanisms involved in the positive and negative selection of T cells., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

B-cell-activating factor of the TNF family (BAFF)/BLyS contributes to B-cell homeostasis and function in the periphery. BAFF is expressed as a membrane-bound protein or released by proteolytic cleavage, but the functional importance of this processing event is poorly understood. Mice expressing BAFF with a mutated furin consensus cleavage site, i.e. furin-mutant BAFF (fmBAFF), were not different from BAFF-deficient mice with regard to their B-cell populations and responses to immunization. It is however noteworthy that an alternative processing event releases some soluble BAFF in fmBAFF mice. Mild overexpression (∼ 5-fold) of fmBAFF alone generated intermediate levels of B cells without improving humoral responses to immunization. Processed BAFF was however important for B-cell homeostasis, as peripheral B-cell populations and antibody responses were readily restored by administration of soluble BAFF trimers in BAFF-deficient mice. However, the rescue of CD23 expression in B cells of BAFF-deficient mice required both soluble BAFF trimers and fmBAFF, or a polymeric form of soluble BAFF (BAFF 60-mer). These results point to a predominant role of processed BAFF for B-cell homeostasis and function, and indicate possible accessory roles for membrane-bound BAFF., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.

B-cell development up to the immature B-cell stage takes place in the bone marrow, while final maturation into mature B cells occurs in the spleen. During differentiation, the precursor and immature B cells have to pass several checkpoints, including those in which they are censored for being auto-reactive, and therefore being potentially dangerous. Numerous studies have shown that the immature B-cell stage in the bone marrow and the transitional B-cell stages in the spleen comprise distinct checkpoints at which auto-reactivity is censored. Recently, evidence has been provided that the large pre-BII stage in the bone marrow, at which the pre-BCR is expressed, is yet another B-cell tolerance checkpoint. Here, we review these findings and speculate on directions for possible further experimentation.

During fetal life, CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch development in mice. In adult animals, CD4(+)CD3(-) cells are found in low numbers in lymphoid organs. Whether adult CD4(+)CD3(-) cells are LTi cells and are generated and maintained through cytokine signals has not been directly addressed. In this study we show that adult CD4(+)CD3(-) cells adoptively transferred into neonatal CXCR5(-/-) mice induced the formation of intestinal lymphoid tissues, demonstrating for the first time their bona fide LTi function. Increasing IL-7 availability in wild-type mice either by IL-7 transgene expression or treatment with IL-7/anti-IL-7 complexes increased adult LTi cell numbers through de novo generation from bone marrow cells and increased the survival and proliferation of LTi cells. Our observations demonstrate that adult CD4(+)lineage(-) cells are LTi cells and that the availability of IL-7 determines the size of the adult LTi cell pool.

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools.

Between 10 and 20% of the peripheral gammadelta T cells express cytoplasmic TCR-beta proteins, but whether such TCR-beta chains can partake in alphabeta T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3epsilon-deficient mice with Pax5-TCR-beta deficient proB cells expressing, via a retroviral vector, TCR-beta chains from either peripheral gammadelta or alphabeta T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (<15x10(6) cells), contained few gammadelta T but no alphabeta T cells. In contrast, thymi from mice receiving proB cells containing gammadelta or alphabeta T-cell-derived TCR-beta chains contained 80-130x10(6) cells, and showed a normal CD4, CD8 and alphabeta TCR expression pattern. However, regardless of the source of TCR-beta chain, reconstituted mice rapidly showed signs of autoimmunity dying 5-15 wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-beta chains from gammadelta T cells can efficiently take part in alphabeta T-cell development. The implications of these findings for gammadelta T-cell development will be discussed.

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.

T cell homeostasis is essential for the functioning of the vertebrate immune system, but the intracellular signals required for T cell homeostasis are largely unknown. We here report that the WD-repeat protein family member coronin-1, encoded by the gene Coro1a, is essential in the mouse for T cell survival through its promotion of Ca2+ mobilization from intracellular stores. Upon T cell receptor triggering, coronin-1 was essential for the generation of inositol-1,4,5-trisphosphate from phosphatidylinositol-4,5-bisphosphate. The absence of coronin-1, although it did not affect T cell development, resulted in a profound defect in Ca2+ mobilization, interleukin-2 production, T cell proliferation and T cell survival. We conclude that coronin-1, through activation of Ca2+ release from intracellular stores, is an essential regulator of peripheral lymphocyte survival.

OBF1, also known as Bob.1 or OCA-B, is a B lymphocyte-specific transcription factor which coactivates Oct1 and Oct2 on B cell specific promoters. So far, the function of OBF1 has been mainly identified in late stage B cell populations. The central defect of OBF1 deficient mice is a severely reduced immune response to T cell-dependent antigens and a lack of germinal center formation in the spleen. Relatively little is known about a potential function of OBF1 in developing B cells. Here we have generated transgenic mice overexpressing OBF1 in B cells under the control of the immunoglobulin heavy chain promoter and enhancer. Surprisingly, these mice have greatly reduced numbers of follicular B cells in the periphery and have a compromised immune response. Furthermore, B cell differentiation is impaired at an early stage in the bone marrow: a first block is observed during B cell commitment and a second differentiation block is seen at the large preB2 cell stage. The cells that succeed to escape the block and to differentiate into mature B cells have post-translationally downregulated the expression of transgene, indicating that expression of OBF1 beyond the normal level early in B cell development is deleterious. Transcriptome analysis identified genes deregulated in these mice and Id2 and Id3, two known negative regulators of B cell differentiation, were found to be upregulated in the EPLM and preB cells of the transgenic mice. Furthermore, the Id2 and Id3 promoters contain octamer-like sites, to which OBF1 can bind. These results provide evidence that tight regulation of OBF1 expression in early B cells is essential to allow efficient B lymphocyte differentiation.

Interleukin 7 (IL-7) plays a crucial role in adult lymphopoiesis, while in fetal life its effect can be partially compensated by TSLP. Whether adult hematopoietic progenitor cells are unresponsive to TSLP or whether TSLP is less available in adult microenvironments is still a matter of debate. Here, we show that increased TSLP availability through transgene (Tg) expression fully restored lymphopoiesis in IL-7-deficient mice: it rescued B-cell development, increased thymic and splenic cellularities, and restored double-negative (DN) thymocytes, alphabeta and gammadelta T-cell generation, and all peripheral lymphoid compartments. Analysis of bone marrow chimeras demonstrated that hematopoietic progenitor cells from adult wild-type mice efficiently differentiated toward B- and T-cell lineages in lethally irradiated IL-7 deficient mice provided TSLP Tg was expressed in these mice. In vitro, TSLP promoted the differentiation of uncommitted adult bone marrow progenitors toward B and T lineages and the further differentiation of DN1 and DN2 thymocytes. Altogether, our results show that adult hematopoietic cells are TSLP responsive and that TSLP can sustain long-term adult lymphopoiesis.

Analysis of hematopoietic development has for decades been central to understanding lineage diversification. Some models consider hematopoietic commitment to be random, and branching lineage maps often include an early myeloid or lymphoid bifurcation. However, the existence of joint lymphoid or myeloid intermediate progenitors argues against both. One of us earlier proposed the sequential determination (SD) model, which features a limited and stepwise set of binary choices across the full hematopoietic spectrum. This model arose from observations that hematopoietic progenitors show preferences for particular associations of lineage potentials--indicating that these linked fates are neighbours developmentally. An updated SD model complemented by several recently recognized processes--spatiotemporal fluctuations in transcription factor concentrations, asymmetric cell division, and Notch signalling--still offers a sound summary of hematopoiesis.

Haematopoietic stem cells (HSCs), a very rare cell type in the bone marrow, are responsible for the life-long production of all cells of the blood including T and B cells. Until recently, it was thought that the differentiation of HSCs into the various haematopoietic cells was rather hierarchical in that differentiation along a given lineage was associated with a progressive loss of potential to give rise to other blood cell lineages. The recent development of very sensitive and quantitative in vitro assays, together with the identification of new progenitor subpopulations, has challenged this idea. Thus, lymphocyte progenitors can be shown to keep their developmental potential to give rise to myeloid, dendritic and NK cells until just prior to their final commitment stage. Here we review these new findings and concepts.

The nature and lineage potential, particularly that for B cells, of thymus settling progenitors (TSP) in the adult mouse has been the subject of considerable debate. Lack of B cell potential would suggest pre-thymic, whereas its presence would suggest intra-thymic loss of B cell potential. Using limiting dilution analysis (LDA) in vitro and transfer experiments in vivo, we show that the B cell potential of TSP is critically dependent on mouse age, reaching a maximum of about 1 in 20 cells at birth, decreasing 50-fold in adult mice. Cells with a TSP phenotype can be found in the neonatal blood. Furthermore, using LDA, we show that Notch ligand signaling of TSP results in the loss of B cell potential with a half-life of approximately 12 h. Taken together, these results indicate that loss of B cell potential by TSP is an intra-thymic event and highlight the developmental pressure acting on the immune system to rapidly colonize primary lymphoid organs with functional progenitors. This critical time coincides with birth in the mouse. In the adult mouse, we estimate than only about 5 TSP cells/day would be required to maintain steady-state thymopoiesis.

In pre-Talpha (pTalpha) gene-deleted mice, the positively selectable CD4+ CD8+ double-positive thymocyte pool is only 1% that in wild-type mice. Consequently, their peripheral T cell compartment is severely lymphopenic with a concomitant increase in proportion of CD25+ FoxP3+ regulatory T cells. Using mixed bone marrow chimeras, where thymic output was 1% normal, the pTalpha(-/-) peripheral T cell phenotype could be reproduced with normal cells. In the pTalpha(-/-) thymus and peripheral lymphoid organs, FoxP3+ CD4+ cells were enriched. Parabiosis experiments showed that many pTalpha(-/-) CD4+ single-positive thymocytes represented recirculating peripheral T cells. Therefore, the enrichment of FoxP3+ CD4+ single-positive thymocytes was not solely due to increased thymic production. Thus, the pTalpha(-/-) mouse serves as a model system with which to study the consequences of chronic decreased thymic T cell production on the physiology of the peripheral T cell compartment.

NK cells constitute an essential element of the innate immune system; however, the cellular and molecular mechanisms that guide their early development are still poorly understood. Here, we demonstrate that in addition to its known crucial role in T cell development, Notch signaling can also be involved in NK cell development. Thus, upon co-culture on OP9 stroma expressing the Notch ligand Delta-like 1 (OP9-DL1), Pax5-deficient pro-B cells, which have multi-lineage potential, efficiently differentiate into T and NK cells. Upon DL-1 signaling, Pax5-deficient pro-B cells down-regulate both surface CD93 expression and transcripts for B cell-specific genes and concomitantly up-regulate T lineage gene transcripts. Subsequent transfer of DL-1-signaled Pax5-deficient pro-B cells onto OP9 stroma in the presence of IL-2 leads to their efficient differentiation into NK1.1(+), functional NK cells. Moreover, bone marrow early progenitor with lymphoid and myeloid differentiation potential (EPLM), which we have previously described as the normal in vivo-equivalent of Pax5-deficient pro-B cells, also gain the ability to differentiate into effector NK cells following transient DL1 Notch-mediated signaling. The potential involvement of Notch signaling in the generation of the NK cell repertoire in vivo is discussed.