Mammalian VPS45 orchestrates trafficking through the endosomal system.

Vacuolar protein sorting 45 homolog (VPS45), a member of the Sec1/Munc18 (SM) family, has been implicated in the regulation of endosomal trafficking. VPS45 deficiency in human patients results in congenital neutropenia, bone marrow fibrosis, and extramedullary renal hematopoiesis. Detailed mechanisms of the VPS45 function are unknown. Here, we show an essential role of mammalian VPS45 in maintaining the intracellular organization of endolysosomal vesicles and promoting recycling of cell-surface receptors. Loss of VPS45 causes defective Rab5-to-Rab7 conversion resulting in trapping of cargos in early endosomes and impaired delivery to lysosomes. In this context, we demonstrate aberrant trafficking of the granulocyte colony-stimulating factor receptor in the absence of VPS45. Furthermore, we find that lack of VPS45 in mice is not compatible with embryonic development. Thus, we identify mammalian VPS45 as a critical regulator of trafficking through the endosomal system and early embryogenesis of mice.

miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity.

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.

miR-191 modulates B-cell development and targets transcription factors E2A, Foxp1, and Egr1.

The interdependence of posttranscriptional gene regulation via miRNA and transcriptional regulatory networks in lymphocyte development is poorly understood. Here, we identified miR-191 as direct upstream modulator of a transcriptional module comprising the transcription factors Foxp1, E2A, and Egr1. Deletion as well as ectopic expression of miR-191 resulted in developmental arrest in B lineage cells, indicating that fine tuning of the combined expression levels of Foxp1, E2A, and Egr1, which in turn control somatic recombination and cytokine-driven expansion, constitutes a prerequisite for efficient B-cell development. In conclusion, we propose that miR-191 acts as a rheostat in B-cell development by fine tuning a key transcriptional program.

T Cell Development by the Numbers.

T cells are continually generated in the thymus in a highly dynamic process comprising discrete steps of lineage commitment, T cell receptor (TCR) gene rearrangement, and selection. These steps are linked to distinct rates of proliferation, survival, and cell death, but a quantitative picture of T cell development is only beginning to emerge. Here we summarize recent technical advances, including genetic fate mapping, barcoding, and molecular timers, that have allowed the implementation of computational models to quantify developmental dynamics in the thymus. Coupling new techniques with mathematical models has recently resulted in the emergence of new paradigms in early hematopoiesis and might similarly open new perspectives on T cell development.

A Rare Case of Activated Phosphoinositide 3-Kinase Delta Syndrome (APDS) Presenting With Hemophagocytosis Complicated With Hodgkin Lymphoma.

Gain of function mutations in the p110δ catalytic subunit of the phosphatidylinositol-3-OH kinase (PIK3CD) classified as activated phosphoinositide 3-kinase delta syndrome (APDS) are the cause of a primary immunodeficiency characterized by recurrent sinopulmonary infections, and lymphoproliferation. Previously, autoimmunity and Epstein-Barr virus-related B-cell lymphoma have been documented for patients with APDS; here, we present a case that extends the picture, as the patient shows the full diagnostic criteria of hemophagocytic lymphohistiocytosis at 6 months of age. He experienced Hodgkin lymphoma as a 2.5-year-old baby. Next-generation sequencing returned a de novo heterozygous missense variant in PIK3CD (LRG_191t1: c.3061G>A; p.Glu1021Lys), confirming the primary immunodeficiency. After 2 courses of ifosfamide, cisplatin, and etoposide combined with brentuximab, the patient successfully underwent allogeneic hematopoietic stem cell transplantation from his HLA full matched sister, and he has been well for 18 months after that. The hematologist treating Hodgkin lymphoma and/or hemophagocytic lymphohistiocytosis should be vigilant about the possible underlying immune deficiency, and they should consider APDS in their differential diagnosis.

Genetic Deficiency and Biochemical Inhibition of ITK Affect Human Th17, Treg, and Innate Lymphoid Cells.

PURPOSE: Interleukin-2-inducible T cell kinase (ITK) is an important mediator of T cell receptor signaling. Loss of function mutations in ITK results in hypogammaglobulinemia and CD4+ T cell loss in humans, and the patients often present with EBV-associated B cell lymphoproliferative syndrome. Itk-deficient mice show loss of T cell naivety, impaired cytolytic activity of CD8+ T cells, and defects in CD4+ T cell lineage choice decisions. In mice, Itk mutations were shown to affect Th17-Treg lineage choice in favor of the latter. In this study, we explored whether human ITK reciprocally regulates Th17-Treg balance as its murine ortholog. METHODS: Whole Exome Sequencing was used to identify the mutation. ITK-deficient peripheral blood lymphocytes were characterized by FACSAria III-based flow cytometric assays with respect to proliferation, apoptosis, cytokine production, and innate lymphoid cell (ILC) frequency. Sorted T cells from healthy donors were exposed to ibrutinib, an irreversible ITK inhibitor, to assess ITK's contribution to Th17 and Treg cell generation and functions. RESULTS: In this study, we report a child with a novel ITK mutation who showed impaired CD3/CD28 induced proliferation in T cells. ITK-mutant cells were more apoptotic irrespective of TCR activation. More importantly, T cells produced less Th17-associated cytokines IL-17A, IL-22, and GM-CSF. Conversely, Th1-associated IFN-γ production was increased. An irreversible inhibitor of ITK, ibrutinib, blocked ex vivo Th17 generation and IL-17A production, conversely augmented FOXP3 expression only at low doses in Treg cultures. Finally, we analyzed peripheral ILC populations and observed a relative decrease in ILC2 and ILC3 frequency in our ITK-deficient patient. CONCLUSIONS: To our knowledge, this is the first report showing that both genetic and chemical inhibition of ITK result in reduced Th17 generation and function in humans. We also report, for the first time, a reduction in ILC2 and ILC3 populations in an ITK-deficient human patient.

Limited niche availability suppresses murine intrathymic dendritic-cell development from noncommitted progenitors.

The origins of dendritic cells (DCs) and other myeloid cells in the thymus have remained controversial. In this study, we assessed developmental relationships between thymic dendritic cells and thymocytes, employing retrovirus-based cellular barcoding and reporter mice, as well as intrathymic transfers coupled with DC depletion. We demonstrated that a subset of early T-lineage progenitors expressed CX3CR1, a bona fide marker for DC progenitors. However, intrathymic transfers into nonmanipulated mice, as well as retroviral barcoding, indicated that thymic dendritic cells and thymocytes were largely of distinct developmental origin. In contrast, intrathymic transfers after in vivo depletion of DCs resulted in intrathymic development of non-T-lineage cells. In conclusion, our data support a model in which the adoption of T-lineage fate by noncommitted progenitors at steady state is enforced by signals from the thymic microenvironment unless niches promoting alternative lineage fates become available.

Responsiveness of Developing T Cells to IL-7 Signals Is Sustained by miR-17∼92.

miRNAs regulate a large variety of developmental processes including development of the immune system. T cell development is tightly controlled through the interplay of transcriptional programs and cytokine-mediated signals. However, the role of individual miRNAs in this process remains largely elusive. In this study, we demonstrated that hematopoietic cell-specific loss of miR-17∼92, a cluster of six miRNAs implicated in B and T lineage leukemogenesis, resulted in profound defects in T cell development both at the level of prethymic T cell progenitors as well as intrathymically. We identified reduced surface expression of IL-7R and concomitant limited responsiveness to IL-7 signals as a common mechanism resulting in reduced cell survival of common lymphoid progenitors and thymocytes at the double-negative to double-positive transition. In conclusion, we identified miR-17∼92 as a critical modulator of multiple stages of T cell development.

Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells.

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

Immunoglobulins drive terminal maturation of splenic dendritic cells.

Nature and physiological status of antigen-presenting cells, such as dendritic cells DCs, are decisive for the immune reactions elicited. Multiple factors and cell interactions have been described that affect maturation of DCs. Here, we show that DCs arising in the absence of immunoglobulins (Ig) in vivo are impaired in cross-presentation of soluble antigen. This deficiency was due to aberrant cellular targeting of antigen to lysosomes and its rapid degradation. Function of DCs could be restored by transfer of Ig irrespective of antigen specificity and isotype. Modulation of cross-presentation by Ig was inhibited by coapplication of mannan and, thus, likely to be mediated by C-type lectin receptors. This unexpected dependency of splenic DCs on Ig to cross-present antigen provides insights into the interplay between cellular and humoral immunity and the immunomodulatory capacity of Ig.

B-cell modulation of dendritic-cell function: signals from the far side.

An appropriate immune response against a specific pathogen requires finely orchestrated interactions between the various cell populations within the immune system. At the same time, immunological tolerance to self must be maintained. DCs play an essential role in achieving these dual requisites. They coordinate adaptive immunity by integrating signals directly emanating from both infectious agents and cells of the immune system. Many such signals, especially those from innate cells and T cells, have been extensively characterized. In contrast, little is known about how B cells modulate function of DCs. B cells produce a variety of cytokines, including IL-10 and IL-6, which are known to influence DC function. In addition, Igs constitute the major secretory products of terminally differentiated B cells (plasma cells). DCs express various types of receptors for binding Ig, such as Fc receptors and C-type lectin receptors. In accordance, Igs can regulate DC function depending on the receptors engaged. Here, we review the emerging immunomodulatory role of cytokines and Ig secreted by B cells. We discuss the evidence for how these B-cell-derived factors may shape the adaptive immune response by directly acting on DCs.

Human IL-21 and IL-21R deficiencies: two novel entities of primary immunodeficiency.

PURPOSE OF REVIEW: This review highlights the recent identification of human interleukin-21 (IL-21) and interleukin-21 receptor (IL-21R) deficiencies as novel entities of primary immunodeficiency. RECENT FINDINGS: We recently described the first patients with IL-21R deficiency who had cryptosporidial infections associated with chronic cholangitis and liver disease. All IL-21R-deficient patients suffered from recurrent respiratory tract infections. Immunological work-up revealed impaired B cell proliferation and immunoglobulin class-switch, reduced T cell effector functions, and variable natural killer cell dysfunctions. Recently, these findings have been extended by the discovery of one patient with a mutation in the IL21 gene. This patient predominantly manifested with very early onset inflammatory bowel disease and recurrent respiratory infections. Laboratory examination showed reduced circulating B cells and impaired B cell class-switch. SUMMARY: Human IL-21 and IL-21R deficiencies cause severe, primary immunodeficiency reminiscent of common variable immunodeficiency. Early diagnosis is critical to prevent life-threatening complications, such as secondary liver failure. In view of the critical role of IL-21 in controlling immune homeostasis, early hematopoietic stem cell transplantation might be considered as therapeutic intervention in affected children.

The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

ICOS-dependent stimulation of NKT cells by marginal zone B cells.

Marginal zone (MZ) B cells express high levels of CD1d molecules. In accordance, MZ B cells, like splenic conventional DCs (cDCs), efficiently trigger NKT-cell proliferation. Importantly, MZ B cells exclusively induced production of IL-4 and IL-13 by such cells whereas cDCs induced robust production of mainly IFN-γ. NKT-cell proliferation, IL-4 and IL-13 production induced by MZ B cells were dependent on ICOS/ICOS ligand interaction while IFN-γ and IL-17 induction by cDCs required glucocorticoid-induced TNF receptor/glucocorticoid-induced TNF receptor ligand interplay. Our data illustrate that both MZ B cells and cDCs act as efficient APCs for NKT cells and might differentially influence the quality of the subsequent immune response.

In vivo RNAi-mediated silencing of TAK1 decreases inflammatory Th1 and Th17 cells through targeting of myeloid cells.

Cells from the mononuclear phagocyte system (MPS) act as systemic and local amplifiers that contribute to the progression of chronic inflammatory disorders. Transforming growth factor-ß-activated kinase 1 (TAK1) is a pivotal upstream mitogen-activated protein kinase-kinase-kinase acting as a mediator of cytokine expression. It remains critical to determine in vivo the implication of TAK1 in controlling the innate immune system. Here, we describe a vehicle tailored to selectively deliver siRNAs into MPS cells after intravenous administration, and validate in vivo the potential of the RNAi-mediated TAK1 knock down for immunomodulation. In a mouse model of immune-mediated inflammatory disorder, we show that anti-TAK1 siRNA lipoplexes efficiently alleviate inflammation, severely impair the downstream c-Jun N-terminal kinase and nuclear factor-κB signaling pathways, and decrease the expression of proinflammatory mediators. Importantly, the systemic TAK1 gene silencing decreases the frequency of Th1 and Th17 cells, both mediating autoimmunity in experimental arthritis, demonstrating the immunomodulatory potential of TAK1. Finally, in vitro inhibition of TAK1 in myeloid cells decreases interferon-γ-producing T cells, suggesting that a delivery system able to target MPS cells and to silence TAK1 impacts on pathogenic T effector cells in autoimmunity.

Unblinding the watchmaker: cancer treatment and drug design in the face of evolutionary pressure.

INTRODUCTION: Death due to cancer is mostly associated with therapy ineffectiveness, i.e. tumor cells no longer responding to treatment. The underlying dynamics that facilitate this mutational escape from selective pressure are well studied in several other fields and several interesting approaches exist to combat this phenomenon, for example in the context of antibiotic-resistance in bacteria. AREAS COVERED: Ninety percent of all cancer-related deaths are associated with treatment failure. Here, we discuss the common treatment modalities and prior attempts to overcome acquired resistance to therapy. The underlying molecular mechanisms are discussed and the implications of emerging resistance in other systems, such as bacteria, are discussed in the context of cancer. EXPERT OPINION: Reevaluating emerging therapy resistance in tumors as an evolutionary mechanism to survive in a rapidly and drastically altering fitness landscape leads to novel treatment strategies and distinct requirements for new drugs. Here, we propose a scheme of considerations that need to be applied prior to the discovery of novel therapeutic drugs.

Temporal and spatial resolution of type I and III interferon responses in vivo.

Although the action of interferons (IFNs) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern of IFN-induced genes in vivo. We created BAC transgenic mice expressing firefly luciferase under transcriptional control of the Mx2 gene promoter. Expression of the reporter with regard to onset and kinetics of induction parallels that of Mx2 and is thus a hallmark for the host response. Substantial constitutive expression of the reporter gene was observed in the liver and most other tissues of transgenic mice, whereas this expression was strongly reduced in animals lacking functional type I IFN receptors. As expected, the reporter gene was induced not only in response to type I (alpha and beta) and type III (lambda) IFNs but also in response to a variety of IFN inducers such as double-stranded RNA, lipopolysaccharide (LPS), and viruses. In vivo IFN subtypes show clear differences with respect to their kinetics of action and to their spatial activation pattern: while the type I IFN response was strong in liver, spleen, and kidney, type III IFN reactivity was most prominent in organs with mucosal surfaces. Infection of reporter mice with virus strains that differ in their pathogenicity shows that the IFN response is significantly altered in the strength of IFN action at sites which are not primarily infected as well as by the onset and duration of gene induction.

Novel reporter mouse reveals constitutive and inflammatory expression of IFN-beta in vivo.

Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-beta as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-beta gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-beta following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-beta is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-beta under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

Absence of IFN-beta impairs antigen presentation capacity of splenic dendritic cells via down-regulation of heat shock protein 70.

Type I IFNs play a key role in linking the innate and adaptive arms of the immune system. Although produced rapidly in response to pathogens, IFNs are also produced at low levels in the absence of infection. In the present study, we demonstrate that constitutively produced IFNs are necessary in vivo to maintain dendritic cells in an "Ag presentation-competent" state. Conventional dendritic cells (cDCs) isolated from spleens of IFN-beta or IFNAR-deficient mice exhibit a highly impaired ability to present Ag and activate naive T cells. Microarray analysis of mRNA isolated from IFN-beta(-/-) and IFNAR(-/-) cDCs revealed diminished expression of two genes that encoded members of the heat shock protein 70 (Hsp70) family. Consistent with this observation, pharmacological inhibition of Hsp70 in cDCs from wild-type mice impaired their T cell stimulatory capacity. Similarly, the Ag presentation ability of splenic cDCs isolated from Hsp70.1/3(-/-) mice was also severely impaired in comparison to wild-type cDCs. Thus, constitutive IFN-beta expression regulates Hsp70 levels to help maintain dendritic cells in a competent state for efficient priming of effector T cells in vivo.

Listeria monocytogenes induces T cell receptor unresponsiveness through pore-forming toxin listeriolysin O.

BACKGROUND: The success of many pathogens relies on their ability to circumvent the innate and adaptive immune defenses. How bacterial pathogens subvert adaptive immune defenses is not clear. Cholesterol-dependent cytolysins (CDCs) represent an expansive family of homologous pore-forming toxins that are produced by more than 20 gram-positive bacterial species. Listeriolysin O (LLO), a prototype CDC, is the main virulence factor of Listeria monocytogenes. METHODS: We employed flow cytometric and microarray techniques to analyze the effect of LLO on T cell activation in vitro and in vivo. RESULTS: In vivo and in vitro proliferation of CD4(+) T cells upon T cell receptor (TCR) activation was highly diminished in the presence of LLO or wild-type L. monocytogenes but not in the presence of LLO-deficient L. monocytogenes. This block in T cell proliferation was specific to T cell activation via the TCR and not by phorbol 12-myristate 13-acetate-ionomycin, which bypasses the proximal TCR signaling event. The results of microarray analysis suggest that LLO-induced T cell unresponsiveness is due to the induction of a calcium-nuclear factor of activated T cells-dependent transcriptional program that drives the expression of negative regulators of TCR signaling. CONCLUSION: These findings provide important insights into how bacterial toxins silence adaptive immune responses and thus enable prolonged survival of the pathogen in the host.