PI3-Kinase-γ Has a Distinct and Essential Role in Lung-Specific Dendritic Cell Development.

Development of dendritic cells (DCs) commences in the bone marrow, from where pre-DCs migrate to peripheral organs to differentiate into mature DCs in situ. However, the factors that regulate organ-specific differentiation to give rise to tissue-specific DC subsets remain unclear. Here we show that the Ras-PI3Kγ-Akt-mTOR signaling axis acted downstream of FLT3L signaling and was required for development of lung CD103(+) DCs and, to a smaller extent, for lung CD11b(+) DCs, but not related DC populations in other non-lymphoid organs. Furthermore, we show that in lymphoid organs such as the spleen, DCs depended on a similar signaling network to respond to FLT3 ligand with overlapping and partially redundant roles for kinases PI3Kγ and PI3Kδ. Thus we identified PI3Kγ as an essential organ-specific regulator of lung DC development and discovered a signaling network regulating tissue-specific DC development mediated by FLT3.

Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors.

Single-cell RNA sequencing is a powerful technology for assessing heterogeneity within defined cell populations. Here, we describe the heterogeneity of a B220+CD117intCD19-NK1.1- uncommitted hematopoietic progenitor having combined lymphoid and myeloid potential. Phenotypic and functional assays revealed four subpopulations within the progenitor with distinct lineage developmental potentials. Among them, the Ly6D+SiglecH-CD11c- fraction was lymphoid-restricted exhibiting strong B-cell potential, whereas the Ly6D-SiglecH-CD11c- fraction showed mixed lympho-myeloid potential. Single-cell RNA sequencing of these subsets revealed that the latter population comprised a mixture of cells with distinct lymphoid and myeloid transcriptional signatures and identified a subgroup as the potential precursor of Ly6D+SiglecH-CD11c- Subsequent functional assays confirmed that B220+CD117intCD19-NK1.1- single cells are, with rare exceptions, not bipotent for lymphoid and myeloid lineages. A B-cell priming gradient was observed within the Ly6D+SiglecH-CD11c- subset and we propose a herein newly identified subgroup as the direct precursor of the first B-cell committed stage. Therefore, the apparent multipotency of B220+CD117intCD19-NK1.1- progenitors results from underlying heterogeneity at the single-cell level and highlights the validity of single-cell transcriptomics for resolving cellular heterogeneity and developmental relationships among hematopoietic progenitors.

Regulation of T cell survival through coronin-1-mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor triggering.

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.

Permissive roles of cytokines interleukin-7 and Flt3 ligand in mouse B-cell lineage commitment.

Hematopoietic cells are continuously generated throughout life from hematopoietic stem cells, thus making hematopoiesis a favorable system to study developmental cell lineage commitment. The main factors incorporating environmental signals to developing hematopoietic cells are cytokines, which regulate commitment of hematopoietic progenitors to the different blood lineages by acting either in an instructive or a permissive manner. Fms-like tyrosine kinase-3 (Flt3) ligand (FL) and Interleukin-7 (IL-7) are cytokines pivotal for B-cell development, as manifested by the severely compromised B-cell development in their absence. However, their precise role in regulating B-cell commitment has been the subject of debate. In the present study we assessed the rescue of B-cell commitment in mice lacking IL-7 but simultaneously overexpressing FL. Results obtained demonstrate that FL overexpression in IL-7-deficient mice rescues B-cell commitment, resulting in significant Ebf1 and Pax5 expression in Ly6D+CD135+CD127+CD19- precursors and subsequent generation of normal numbers of CD19+ B-cell progenitors, therefore indicating that IL-7 can be dispensable for commitment to the B-cell lineage. Further analysis of Ly6D+CD135+CD127+CD19- progenitors in IL-7- or FL-deficient mice overexpressing Bcl2, as well as in IL-7 transgenic mice suggests that both FL and IL-7 regulate B-cell commitment in a permissive manner: FL by inducing proliferation of Ly6D+CD135+CD127+CD19- progenitors and IL-7 by providing survival signals to these progenitors.

Pro-B cells propagated in stromal cell-free cultures reconstitute functional B-cell compartments in immunodeficient mice.

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 IgG1 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.

Flt3 Ligand Regulates the Development of Innate Lymphoid Cells in Fetal and Adult Mice.

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 α4ß7(-) and α4ß7(+) 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.

Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation.

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1ß stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

The Cytokine Flt3-Ligand in Normal and Malignant Hematopoiesis.

The cytokine Fms-like tyrosine kinase 3 ligand (FL) is an important regulator of hematopoiesis. Its receptor, Flt3, is expressed on myeloid, lymphoid and dendritic cell progenitors and is considered an important growth and differentiation factor for several hematopoietic lineages. Activating mutations of Flt3 are frequently found in acute myeloid leukemia (AML) patients and associated with a poor clinical prognosis. In the present review we provide an overview of our current knowledge on the role of FL in the generation of blood cell lineages. We examine recent studies on Flt3 expression by hematopoietic stem cells and its potential instructive action at early stages of hematopoiesis. In addition, we review current findings on the role of mutated FLT3 in leukemia and the development of FLT3 inhibitors for therapeutic use to treat AML. The importance of mouse models in elucidating the role of Flt3-ligand in normal and malignant hematopoiesis is discussed.

De novo DNA Methyltransferases Dnmt3a and Dnmt3b regulate the onset of Igκ light chain rearrangement during early B-cell development.

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.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

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 TCRß 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.

A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.

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.

A feeder-free differentiation system identifies autonomously proliferating B cell precursors in human bone marrow.

The peripheral B cell compartment is maintained by homeostatic proliferation and through replenishment by bone marrow precursors. Because hematopoietic stem cells cycle at a slow rate, replenishment must involve replication of precursor B cells. To study proliferation of early human B cell progenitors, we established a feeder cell-free in vitro system allowing the development of B cells from CD34(+) hematopoietic stem cells up to the stage of immature IgM(+) B cells. We found that pro-B and pre-B cells generated in vitro can proliferate autonomously and persist up to 7 wk in culture in the absence of signals induced by exogenously added cytokines. Nevertheless, addition of IL-7 enhanced pre-B cell expansion and inhibited maturation into IgM(+) B cells. The B cell precursor subsets replicating in vitro were highly similar to the bone marrow B cell precursors cycling in vivo. The autonomous proliferation of B cell precursor subsets in vitro and their long-term persistence implies that proliferation during pro-B and pre-B cell stages plays an important role in the homeostasis of the peripheral B cell compartment. Our in vitro culture can be used to study defects in B cell development or in reconstitution of the B cell pool after depletion and chemotherapy.

The key role of IL-7 in lymphopoiesis.

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.

Transcriptional networks in developing and mature B cells.

The development of B cells from haematopoietic stem cells proceeds along a highly ordered, yet flexible, pathway. At multiple steps along this pathway, cells are instructed by transcription factors on how to further differentiate, and several check-points have been identified. These check-points are initial commitment to lymphocytic progenitors, specification of pre-B cells, entry to the peripheral B-cell pool, maturation of B cells and differentiation into plasma cells. At each of these regulatory nodes, there are transcriptional networks that control the outcome, and much progress has recently been made in dissecting these networks. This article reviews our current understanding of this exciting field.

Versatility of stem and progenitor cells and the instructive actions of cytokines on hematopoiesis.

For many years, developing hematopoietic cells have been strictly compartmentalized into a rare population of multi-potent self-renewing hematopoietic stem cells (HSC), multi-potent hematopoietic progenitor cells (MPP) that are undergoing commitment to particular lineage fates, and recognizable precursor cells that mature towards functional blood and immune cells. A single route to each end-cell type is prescribed in the "classical" model for the architecture of hematopoiesis. Recent findings have led to the viewpoint that HSCs and MPPs are more versatile than previously thought. Underlying this are multiple routes to a particular fate and cells having clandestine fate options even when they have progressed some way along a pathway. The primary role of cytokines during hematopoiesis has long been seen to be regulation of the survival and proliferation of developing hematopoietic cells. Some cytokines now clearly have instructive actions on cell-fate decisions. All this leads to a new way of viewing hematopoiesis whereby versatile HSC and MPP are directed towards lineage outcomes via cytokine regulated cell-fate decisions. This means greater flexibility to the shaping of hematopoiesis.

The development of autoimmune features in aging mice is closely associated with alterations of the peripheral CD4⁺ T-cell compartment.

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8-12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4(+) T-cell dependent, since old (40-50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age-related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4(+) T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T-cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T-cell homeostasis may drive the onset of some autoimmune features.

An epigenetic profile of early T-cell development from multipotent progenitors to committed T-cell descendants.

Cellular differentiation of the T-cell branch of the immune system begins with the HSC, which undergoes a series of stages characterized by progressive restriction in multipotency and acquisition of specific lineage identity At the molecular level, the restriction of cell potential, commitment, and differentiation to a specific lineage is achieved through the coordinated control of gene expression and epigenetic mechanisms. Here, we analyzed and compared the gene expression profiles and the genome-wide histone modification marks H3K4me3 (H3 lysine 4 trimethylation) and H3K27me3 (H3 lysine 27 trimethylation) in (i) in vitro propagated HSCs, (ii) in vitro generated and propagated pro-T cells derived from these stem cells, and (iii) double-positive thymocytes derived from these pro-T cells after injection into Rag-deficient mice. The combined analyses of the different datasets in this unique experimental system highlighted the importance of both transcriptional and epigenetic repression in shaping the early phases of T-cell development.

The amount of self-antigen determines the effector function of murine T cells escaping negative selection.

Autoimmune diseases develop when self-specific T cells that escaped negative selection initiate a harmful immune response against self. However, factors, which influence the initiation and progression of an autoimmune response remain incompletely understood. By establishing a double-transgenic BALB/c mouse system in which different amounts of a cell-surface neo-self-antigen are expressed under the CD11c promoter, we demonstrate that antigen dose dramatically influences T-cell tolerance mechanisms. Moderate antigen expression in both hematopoietic and nonhematopoietic cells favors the development of antigen-specific Treg cells and the establishment of a tolerogenic environment. In marked contrast, a high dose of antigen expression results in very stringent negative selection, in poor development of antigen-specific Treg cells and in the early onset of anemia and splenomegaly and the late development of arthritis and high titers of IgG auto Abs. Disease is initiated by autoreactive T cells, which escape negative selection by expressing a second TCR with a different specificity or an altered affinity. Transfer of Ag-specific Treg cells ameliorates the early onset signs of disease but does not prevent the development of long-term chronic pathologies. Altogether, our results suggest that Ag dose directly affects Treg-cell generation and thus, the set-up of T-cell tolerance.

The immunogenicity of antibody aggregates in a novel transgenic mouse model.

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.