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.

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.

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.

HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation.

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1-/- cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.

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.

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.

Author Correction: Human FCHO1 deficiency reveals role for clathrin-mediated endocytosis in development and function of T cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human FCHO1 deficiency reveals role for clathrin-mediated endocytosis in development and function of T cells.

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans.

LAMTOR2 (p14) Controls B Cell Differentiation by Orchestrating Endosomal BCR Trafficking.

B-cell development and function depend on stage-specific signaling through the B-cell antigen receptor (BCR). Signaling and intracellular trafficking of the BCR are connected, but the molecular mechanisms of this link are incompletely understood. Here, we investigated the role of the endosomal adaptor protein and member of the LAMTOR/Ragulator complex LAMTOR2 (p14) in B-cell development. Efficient conditional deletion of LAMTOR2 at the pre-B1 stage using mb1-Cre mice resulted in complete developmental arrest. Deletion of LAMTOR2 using Cd19-Cre mice permitted analysis of residual B cells at later developmental stages, revealing that LAMTOR2 was critical for the generation and activation of mature B lymphocytes. Loss of LAMTOR2 resulted in aberrant BCR signaling due to delayed receptor internalization and endosomal trafficking. In conclusion, we identify LAMTOR2 as critical regulator of BCR trafficking and signaling that is essential for early B-cell development in 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.

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.

MicroRNA-181a/b-1 Is Not Required for Innate γδ NKT Effector Cell Development.

Thymic development of αß T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1-/-mice, which show impaired agonistic T cell selection of invariant αß NKT cells, were crossed to Tcrd-H2BeGFP reporter mice to monitor selection, intra-thymic expansion and differentiation of γδ T cells. We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αß NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes.

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.

Multicongenic fate mapping quantification of dynamics of thymus colonization.

Postnatal T cell development depends on continuous colonization of the thymus by BM-derived T lineage progenitors. Both quantitative parameters and the mechanisms of thymus seeding remain poorly understood. Here, we determined the number of dedicated thymus-seeding progenitor niches (TSPNs) capable of supporting productive T cell development, turnover rates of niche occupancy, and feedback mechanisms. To this end, we established multicongenic fate mapping combined with mathematical modeling to quantitate individual events of thymus colonization. We applied this method to study thymus colonization in CCR7(-/-)CCR9(-/-) (DKO) mice, whose TSPNs are largely unoccupied. We showed that ∼160-200 TSPNs are present in the adult thymus and, on average, 10 of these TSPNs were open for recolonization at steady state. Preconditioning of wild-type mice revealed a similar number of TSPNs, indicating that preconditioning can generate space efficiently for transplanted T cell progenitors. To identify potential cellular feedback loops restricting thymus colonization, we performed serial transfer experiments. These experiments indicated that thymus seeding was directly restricted by the duration of niche occupancy rather than long-range effects, thus challenging current paradigms of thymus colonization.

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.

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.

S/T phosphorylation of DLL1 is required for full ligand activity in vitro but dispensable for DLL1 function in vivo during embryonic patterning and marginal zone B cell development.

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.

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.