Human genetic defects in SRP19 and SRPRA cause severe congenital neutropenia with distinctive proteome changes.

The mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell-based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

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.

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.

Overexpression of Vα14Jα18 TCR promotes development of iNKT cells in the absence of miR-181a/b-1.

Expression of microRNA miR-181a/b-1 is critical for intrathymic development of invariant natural killer T (iNKT) cells. However, the underlying mechanism has remained a matter of debate. On the one hand, growing evidence suggested that miR-181a/b-1 is instrumental in setting T-cell receptor (TCR) signaling threshold and thus permits agonist selection of iNKT cells through high-affinity TCR ligands. On the other hand, alterations in metabolic fitness mediated by miR-181a/b-1-dependent dysregulation of phosphatase and tensin homolog (Pten) have been proposed to cause the iNKT-cell defect in miR-181-a/b-1-deficient mice. To re-assess the hypothesis that modulation of TCR signal strength is the key mechanism by which miR-181a/b-1 controls the development of iNKT cells, we generated miR-181a/b-1-deficient mice expressing elevated levels of a Vα14Jα18 TCRα chain. In these mice, development of iNKT cells was fully restored. Furthermore, both subset distribution of iNKT cells as well as TCR Vß repertoire were independent of the presence of miR-181a/b-1 once a Vα14Jα18 TCRα chain was overexpressed. Finally, levels of Pten protein were similar in Vα14Jα18 transgenic mice irrespective of their miR-181a/b-1 status. Collectively, our data support a model in which miR-181 promotes development of iNKT cells primarily by generating a permissive state for agonist selection with alterations in metabolic fitness possibly constituting a secondary effect.

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.

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.

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.

CCR7-mediated migration in the thymus controls γδ T-cell development.

αß T-cell development and selection proceed while thymocytes successively migrate through distinct regions of the thymus. For γδ T cells, the interplay of intrathymic migration and cell differentiation is less well understood. Here, we crossed C-C chemokine receptor (CCR)7-deficient (Ccr7(-/-) ) and CCR9-deficient mice (Ccr9(-/-) ) to mice with a TcrdH2BeGFP reporter background to investigate the impact of thymic localization on γδ T-cell development. γδ T-cell frequencies and numbers were decreased in CCR7-deficient and increased in CCR9-deficient mice. Transfer of CCR7- or CCR9-deficient BM into irradiated C57BL/6 WT recipients reproduced these phenotypes, pointing toward cell-intrinsic migration defects. Monitoring recent thymic emigrants by intrathymic labeling allowed us to identify decreased thymic γδ T-cell output in CCR7-deficient mice. In vitro, CCR7-deficient precursors showed normal γδ T-cell development. Immunohistology revealed that CCR7 and CCR9 expression was important for γδ T-cell localization within thymic medulla or cortex, respectively. However, γδ T-cell motility was unaltered in CCR7- or CCR9-deficient thymi. Together, our results suggest that proper intrathymic localization is important for normal γδ T-cell development.

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.

Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, gene-specific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression, DNA-binding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells.

Chemokine receptor CX3CR1 promotes dendritic cell development under steady-state conditions.

Expression of CX3CR1 is an attribute of myeloid precursors committed to the monocyte/macrophage (Mφ)/DC lineages and is maintained during all stages of DC differentiation. Nevertheless, the exact role of this molecule during developmental progression of myeloid precursors towards the DC lineage remains elusive. To overcome potential compensatory mechanisms and issues of redundancy, we employed competitive adoptive transfer experiments to assess a possible function of CX3CR1 in DC and monocyte/Mφ differentiation in vivo. We show here that expression of CX3CR1 promotes the generation of DCs and monocytes/Mφ under steady-state conditions and during compensatory expansion after selective depletion of DCs, but not under inflammatory conditions evoked by sub-lethal irradiation. Direct administration of CX3CR1-deficient and CX3CR1-sufficient precursors into the spleen or the thymus resulted in a similar competitive advantage of WT over CX3CR1-deficient precursors as i.v. transfer, suggesting that CX3CR1-mediated survival rather than recruitment to lymphoid organs is critical for DC/Mφ differentiation. In conclusion, our data support the hypothesis that CX3CR1 promotes proper development of myeloid precursors into DCs and monocytes/Mφs under steady-state conditions, possibly by providing survival signals or mediating accessibility to organ-specific niches, rather than acting as a mediator of homing to the spleen or the thymus.

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.

CC chemokine receptor 7 and 9 double-deficient hematopoietic progenitors are severely impaired in seeding the adult thymus.

T-cell development depends on recruitment of bone marrow-derived precursor cells to the thymus via a multistep adhesion cascade involving the chemokine receptor CCR9. However, CCR9 deficiency does not result in complete abrogation of progenitor entry into the adult thymus. Therefore, we tested the hypothesis that additional chemokine/chemokine receptor systems might play a role in this process. To this end, we generated mice deficient in both CCR9 and CCR7. Deficiency in both chemokine receptors resulted in severely reduced numbers of early T-cell progenitors and in near-complete abrogation of thymus reconstitution. Progenitors in bone marrow and peripheral blood remained largely unaffected in CCR7(-/-)CCR9(-/-) mice, and direct intrathymic transfer of precursors from CCR7(-/-)CCR9(-/-) mice as well as single-mutant mice showed that intrathymic differentiation of these precursors remained functional. Thus, our data reveal a previously unrecognized role of CCR7 in progenitor seeding of the adult thymus, which is largely masked by compensatory effects of CCR9 signals. In turn, CCR7 signals can partially compensate for CCR9 signals, thus explaining the rather mild phenotype of CCR9(-/-) mice with respect to progenitor seeding.

Multiple extrathymic precursors contribute to T-cell development with different kinetics.

T-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM). Several extrathymic candidate progenitors have been described that range from multipotent cells to lymphoid cell committed progenitors and even largely T-lineage committed precursors. However, the nature of precursors seeding the thymus under physiologic conditions has remained largely elusive and it is not known whether there is only one physiologic T-cell precursor population or many. Here, we used a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby only minimally altering physiologic precursor ratios to assess the contribution of various extrathymic precursors to T-lineage differentiation. We found that under these conditions multiple precursors, belonging to both multipotent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T-lineage potential. However, differentiation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extrathymic precursors. In conclusion, our data suggest that the thymus functions to impose T-cell fate on any precursor capable of filling the limited number of progenitor niches.

Post-transcriptional control of T-cell development in the thymus.

T-cell development in the thymus is dependent on the continual colonization by bone-marrow derived progenitor cells. Once inside the thymus, progenitors undergo a series of well-defined differentiation events, including lineage commitment, somatic recombination of T-cell receptor (TCR) gene loci, and selection of clones with productively recombined yet non-autoreactive TCRs. Cell-cell interactions, cytokine signals, transcriptional as well as epigenetic programs controlling T-cell development are comparatively well-characterized. In contrast, the contribution of post-transcriptional control and its underlying mechanisms remain largely elusive. Here, we summarize recent advances in our understanding of post-transcriptional regulation of T-cell development, focussing on microRNAs (miRNAs) and RNA-binding proteins (RBPs). We highlight the current challenges, and how they can potentially be overcome with evolving sophisticated methodology to enable a thorough mechanistic understanding and decipher the regulatory networks operating in the gene expression programs of T-cell development.

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.