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Congenital hydrocephalus (CH), characterized by cerebral ventriculomegaly, is one of the most common reasons for pediatric brain surgery. Recent studies have implicated lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) as a candidate CH risk gene, however, TRIM71 variants have not been systematically examined in a large patient cohort or conclusively linked with an OMIM syndrome. Through cross-sectional analysis of the largest assembled cohort of patients with cerebral ventriculomegaly, including neurosurgically-treated CH (totaling 2,697 parent-proband trios and 8,091 total exomes), we identified 13 protein-altering de novo variants (DNVs) in TRIM71 in unrelated children exhibiting variable ventriculomegaly, CH, developmental delay, dysmorphic features, and other structural brain defects including corpus callosum dysgenesis and white matter hypoplasia. Eight unrelated patients were found to harbor arginine variants, including two recurrent missense DNVs, at homologous positions in RPXGV motifs of different NHL domains. Seven additional patients with rare, damaging, unphased or transmitted variants of uncertain significance were also identified. NHL-domain variants of TRIM71 exhibited impaired binding to the canonical TRIM71 target CDKN1A; other variants failed to direct the subcellular localization of TRIM71 to processing bodies. Single-cell transcriptomic analysis of human embryos revealed expression of TRIM71 in early first-trimester neural stem cells of the brain. These data show TRIM71 is essential for human brain morphogenesis and that TRIM71 mutations cause a novel neurodevelopmental syndrome featuring ventriculomegaly and CH., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The consumption of processed food is on the rise leading to huge intake of excess dietary salt, which strongly correlates with development of hypertension, often leading to cardiovascular diseases such as stroke and heart attack, as well as activation of the immune system. The effect of salt on macrophages is especially interesting as they are able to sense high sodium levels in tissues leading to transcriptional changes. In the skin, macrophages were shown to influence lymphatic vessel growth which, in turn, enables the transport of excess salt and thereby prevents the development of high blood pressure. Furthermore, salt storage in the skin has been linked to the onset of pro-inflammatory effector functions of macrophages in pathogen defence. However, there is only little known about the mechanisms which are involved in changing macrophage function to salt exposure. Here, we characterize the response of macrophages to excess salt both in vitro and in vivo. Our results validate and strengthen the notion that macrophages exhibit chemotactic migration in response to salt gradients in vitro. Furthermore, we demonstrate a reduction in phagocytosis and efferocytosis following acute salt challenge in vitro. While acute exposure to a high-salt diet in vivo has a less pronounced impact on macrophage core functions such as phagocytosis, our data indicate that prolonged salt challenge may exert a distinct effect on the function of macrophages. These findings suggest a potential role for excessive salt sensing by macrophages in the manifestation of diseases related to high-salt diets and explicitly highlight the need for in vivo work to decipher the physiologically relevant impact of excess salt on tissue and cell function., (© 2024. The Author(s).)

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming; however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 re-expression increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71's RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation., (© 2023 The Authors.)

Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis., (© 2022 Weier et al.)

Navigation of dendritic cells (DCs) from the site of infection to lymphoid organs is guided by concentration gradients of CCR7 ligands. How cells interpret chemokine gradients and how they couple directional sensing to polarization and persistent chemotaxis has remained largely elusive. Previous experimental systems were limited in the ability to control fast de novo formation of the final gradient slope, long-lasting stability of the gradient and to expose cells to dynamic stimulation. Here, we used a combination of microfluidics and quantitative in vitro live cell imaging to elucidate the chemotactic sensing strategy of DCs. The microfluidic approach allows us to generate soluble gradients with high spatio-temporal precision and to analyze actin dynamics, cell polarization, and persistent directional migration in both static and dynamic environments. We demonstrate that directional persistence of DC migration requires steady-state characteristics of the soluble gradient instead of temporally rising CCL19 concentration, implying that spatial sensing mechanisms control chemotaxis of DCs. Kymograph analysis of actin dynamics revealed that the presence of the CCL19 gradient is essential to stabilize leading edge protrusions in DCs and to determine directionality, since both cytoskeletal polarization and persistent chemotaxis are abrogated in the range of seconds when steady-state gradients are perturbed. In contrast to Dictyostelium amoeba, DCs are unable to decode oscillatory stimulation of soluble chemokine traveling waves into a directional response toward the wave source. These findings are consistent with the notion that DCs do not employ adaptive temporal sensing strategies that discriminate temporally increasing and decreasing chemoattractant concentrations in our setting. Taken together, in our experimental system DCs do not depend on increasing absolute chemokine concentration over time to induce persistent migration and do not integrate oscillatory stimulation. The observed capability of DCs to migrate with high directional persistence in stable gradients but not when subjected to periodic temporal cues, identifies spatial sensing as a key requirement for persistent chemotaxis of DCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Quast, Zölzer, Guu, Alvarez, Küsters, Kiermaier, Kaupp and Kolanus.)

Cationic liposomes are versatile lipid nanocarriers to improve the pharmacological properties of drug payloads. Recent advantages include the application of their intrinsic immunostimulatory effects to enhance immune activation. Herein, we report for the first time the structural effect of cationic lipids in promoting T cell activation and differentiation in vitro. Two types of cationic liposomes R3C14 and R5C14 were prepared from single type of lipids Arg-C3-Clu2C14 or Arg-C5-Clu2C14, which bear arginine head group and ditetradecyl tails but vary in the carbon number of the spacer in between. Murine CD8 or CD4 T cells were pretreated with 50 μM of each type of liposomes for 2 h, followed by stimulation with anti-CD3/CD28 antibodies for 24 h. In comparison to liposome-untreated T cells, R5C14-pretreatment induced a robust T cell activation (IL-2, CD25 + ) and differentiation into effector cells (CD44 high , CD62L low ), whereas R3C14 did not show comparable effect. Furthermore, a weak activation of nuclear factor of activated T cells (NFAT) was detected in Jurkat-Lucia NFAT cells (InvivoGen), suggesting a potential signaling pathway for the liposomal effect. Although R5C14 liposomes did not activate T cells without subsequent CD3/CD28 stimulation, this study implied a recessive effect of some cationic adjuvant in priming T cells to enhance their responsiveness to antigens., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Interferon (IFN)-induced guanosine triphosphate hydrolysing enzymes (GTPases) have been identified as cornerstones of IFN-mediated cell-autonomous defence. Upon IFN stimulation, these GTPases are highly expressed in various host cells, where they orchestrate anti-microbial activities against a diverse range of pathogens such as bacteria, protozoan and viruses. IFN-induced GTPases have been shown to interact with various host pathways and proteins mediating pathogen control via inflammasome activation, destabilising pathogen compartments and membranes, orchestrating destruction via autophagy and the production of reactive oxygen species as well as inhibiting pathogen mobility. In this mini-review, we provide an update on how the IFN-induced GTPases target pathogens and mediate host defence, emphasising findings on protection against bacterial pathogens., (© 2021 The Author(s).)

Three-dimensional (3D) culture bridges and minimizes the gap between in vitro and in vivo states of cells and various 3D culture systems have been developed according to different approaches. However, most of these approaches are either complicated to operate, or costive to scale up. Therefore, a simple method for stem cell spheroid formation and preservation was proposed using poly(D,L-lactic acid) porous thin film (porous nanosheet), which were fabricated by a roll-to-roll gravure coating method combining a solvent etching process. The obtained porous nanosheet was less than 200 nm in thickness and had an average pore area of 6.6 μm 2 with a porosity of 0.887. It offered a semi-adhesive surface for stem cells to form spheroids and maintained the average spheroid diameter below 100 μm for 5 days. In comparison to the spheroids formed in suspension culture, the porous nanosheets improved cell viability and cell division rate, suggesting the better feasibility to be applied as 3D culture scaffolds., Competing Interests: ST was an inventor of patent (PCT/JP2013/056823) of porous nanosheets and collaborating with Nanotheta Co., Ltd., which is holding the patent. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tsai, Li, Torres-Fernández, Weise, Kolanus and Takeoka.)

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71 -deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis., Competing Interests: LT-F held a stipend which was donated by Bayer AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Torres-Fernández, Emich, Port, Mitschka, Wöste, Schneider, Fietz, Oud, Di Persio, Neuhaus, Kliesch, Hölzel, Schorle, Friedrich, Tüttelmann and Kolanus.)

The stem cell-specific RNA-binding protein TRIM71/LIN-41 was the first identified target of the pro-differentiation and tumor suppressor miRNA let-7. TRIM71 has essential functions in embryonic development and a proposed oncogenic role in several cancer types, such as hepatocellular carcinoma. Here, we show that TRIM71 regulates let-7 expression and activity via two independent mechanisms. On the one hand, TRIM71 enhances pre-let-7 degradation through its direct interaction with LIN28 and TUT4, thereby inhibiting let-7 maturation and indirectly promoting the stabilization of let-7 targets. On the other hand, TRIM71 represses the activity of mature let-7 via its RNA-dependent interaction with the RNA-Induced Silencing Complex (RISC) effector protein AGO2. We found that TRIM71 directly binds and stabilizes let-7 targets, suggesting that let-7 activity inhibition occurs on active RISCs. MiRNA enrichment analysis of several transcriptomic datasets from mouse embryonic stem cells and human hepatocellular carcinoma cells suggests that these let-7 regulatory mechanisms shape transcriptomic changes during developmental and oncogenic processes. Altogether, our work reveals a novel role for TRIM71 as a miRNA repressor and sheds light on a dual mechanism of let-7 regulation., (Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function., Competing Interests: The authors have declared that no competing interests exist.

Nonsense-mediated decay (NMD) plays a fundamental role in the degradation of premature termination codon (PTC)-containing transcripts, but also regulates the expression of functional transcripts lacking PTCs, although such 'non-canonical' functions remain ill-defined and require the identification of factors targeting specific mRNAs to the NMD machinery. Our work identifies the stem cell-specific mRNA repressor protein TRIM71 as one of these factors. TRIM71 plays an essential role in embryonic development and is linked to carcinogenesis. For instance, TRIM71 has been correlated with advanced stages and poor prognosis in hepatocellular carcinoma. Our data shows that TRIM71 represses the mRNA of the cell cycle inhibitor and tumor suppressor CDKN1A/p21 and promotes the proliferation of HepG2 tumor cells. CDKN1A specific recognition involves the direct interaction of TRIM71 NHL domain with a structural RNA stem-loop motif within the CDKN1A 3'UTR. Importantly, CDKN1A repression occurs independently of miRNA-mediated silencing. Instead, the NMD factors SMG1, UPF1 and SMG7 assist TRIM71-mediated degradation of CDKN1A mRNA, among other targets. Our data sheds light on TRIM71-mediated target recognition and repression mechanisms and uncovers a role for this stem cell-specific factor and oncogene in non-canonical NMD, revealing the existence of a novel mRNA surveillance mechanism which we have termed the TRIM71/NMD axis., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Insulin plays a central role in regulating metabolic homeostasis and guanine-nucleotide exchange factors of the cytohesin family have been suggested to be involved in insulin signal transduction. The Drosophila homolog of cytohesin-3, steppke, has been shown to be essential for insulin signaling during larval development. However, genetic evidence for the functional importance of cytohesin-3 in mammals is missing. We therefore analyzed the consequences of genetic cytohesin-3-deficiency on insulin signaling and function in young and aged mice, using normal chow or high-fat diet (HFD). Insulin-receptor dependent signaling events are significantly reduced in liver and adipose tissue of young cytohesin-3-deficient mice after insulin-injection, although blood glucose levels and other metabolic parameters remain normal in these animals. Interestingly, however, cytohesin-3-deficient mice showed a reduced age- and HFD-induced weight gain with a significant reduction of body fat compared to wild-type littermates. Furthermore, cytohesin-3-deficient mice on HFD displayed no alterations in energy expenditure, but had an increased lipid excretion instead, as well as a reduced expression of genes essential for bile acid synthesis. Our findings show for the first time that an intact cyth3 locus is required for full insulin signaling in mammals and might constitute a novel therapeutic target for weight reduction.

Interleukin-2 (IL-2) is a key regulator of adaptive immune responses but its regulation is incompletely understood. We previously found that PDL1-dependent signals were pivotal for liver sinusoidal endothelial cell-mediated priming of CD8 T cells, which have a strongly reduced capacity to produce IL-2. Here, we show that the expression of the ARF-like GTPase Arl4d is PD-L1-dependently induced in such LSEC-primed T cells, and is associated with reduced IL-2 secretion and Akt phosphorylation. Conversely, Arl4d-deficient T cells overproduced IL-2 upon stimulation. Arl4d-deficiency in CD8 T cells also enhanced their expansion and effector function during viral infection in vivo. Consistent with their increased IL-2 production, Arl4d-deficient T cells showed enhanced development into KLRG1 + CD127 - short-lived effector cells (SLEC), which is dependent on IL-2 availability. Thus, our data reveal a PD-L1-dependent regulatory circuitry that involves the induction of Arl4d for limiting IL-2 production in T cells.

Type I IFN production of plasmacytoid dendritic cells (pDCs) triggered by TLR-signaling is an essential part of antiviral responses and autoimmune reactions. Although it was well-documented that members of the cytokine signaling (SOCS) family regulate TLR-signaling, the mechanism of how SOCS proteins regulate TLR7-mediated type I IFN production has not been elucidated yet. In this article, we show that TLR7 activation in human pDCs induced the expression of SOCS1 and SOCS3. SOCS1 and SOCS3 strongly suppressed TLR7-mediated type I IFN production. Furthermore, we demonstrated that SOCS1- and SOCS3-bound IFN regulatory factor 7, a pivotal transcription factor of the TLR7 pathway, through the SH2 domain to promote its proteasomal degradation by lysine 48-linked polyubiquitination. Together, our results demonstrate that SOCS1/3-mediated degradation of IFN regulatory factor 7 directly regulates TLR7 signaling and type I IFN production in pDCs. This mechanism might be targeted by therapeutic approaches to either enhance type I IFN production in antiviral treatment or decrease type I IFN production in the treatment of autoimmune diseases., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Nuclear factor kappaB (NF-κB) is a central transcription factor in the immune system and modulates cell survival in response to radiotherapy. Activation of NF-κB was shown to be an early step in the cellular response to ultraviolet A (UVA) and ionizing radiation exposure in human cells. NF-κB activation by the genotoxic stress-dependent sub-pathway after exposure to different radiation qualities had been evaluated to a very limited extent. In addition, the resulting gene expression profile, which shapes the cellular and tissue response, is unknown. Therefore, in this study the activation of NF-κB after exposure to low- and high-linear energy transfer (LET) radiation and the expression of its target genes were analyzed in human embryonic kidney (HEK) cells. The activation of NF-κB via canonical and genotoxic stress-induced pathways was visualized by the cell line HEK-pNF-κB-d2EGFP/Neo L2 carrying the destabilized enhanced green fluorescent protein (d2EGFP) as reporter. The NF-κB-dependent d2EGFP expression after irradiation with X rays and heavy ions was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after irradiation with X rays (significant NF-κB activation for doses >4 Gy) and heavy ions (significant NF-κB activation at doses as low as 1 Gy), it was expected that radiation quality (LET) played an important role in the cellular radiation response. In addition, the relative biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival were compared for heavy ions having a broad LET range (∼0.3-9,674 keV/μm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real-time reverse transcriptase quantitative PCR (RT-qPCR). The maximal RBE for NF-κB activation and cell killing occurred at an LET value of 80 and 175 keV/μm, respectively. There was a dose-dependent increase in expression of NF-κB target genes NF-κB1A and CXCL8. A qPCR array of 84 NF-κB target genes revealed that TNF and a set of CXCL genes (CXCL1, CXCL2, CXCL8, CXCL10), CCL2, VCAM1, CD83, NF-κB1, NF-κB2 and NF-κBIA were strongly upregulated after exposure to X rays and neon ions (LET 92 keV/μm). After heavy-ion irradiations, it was noted that the expression of NF-κB target genes such as chemokines and CD83 was highest at an LET value that coincided with the LET resulting in maximal NF-κB activation, whereas expression of the NF-κB inhibitory gene NFKBIA was induced transiently by all radiation qualities investigated. Taken together, these findings clearly demonstrate that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ∼50-200 keV/μm. The upregulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, CXCL8/IL-8 and TNF) could be important for cell-cell communication among hit as well as nonhit cells (bystander effect).

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Evolution of tumor cell phenotypes promotes heterogeneity and therapy resistance. Here we found that induction of CD73, the enzyme that generates immunosuppressive adenosine, is linked to melanoma phenotype switching. Activating MAPK mutations and growth factors drove CD73 expression, which marked both nascent and full activation of a mesenchymal-like melanoma cell state program. Proinflammatory cytokines like TNFα cooperated with MAPK signaling through the c-Jun/AP-1 transcription factor complex to activate CD73 transcription by binding to an intronic enhancer. In a mouse model of T-cell immunotherapy, CD73 was induced in relapse melanomas, which acquired a mesenchymal-like phenotype. We also detected CD73 upregulation in melanoma patients progressing under adoptive T-cell transfer or immune checkpoint blockade, arguing for an adaptive resistance mechanism. Our work substantiates CD73 as a target to combine with current immunotherapies, but its dynamic regulation suggests limited value of CD73 pretreatment expression as a biomarker to stratify melanoma patients. Cancer Res; 77(17); 4697-709. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Rapidity and specificity are characteristic features of proteolysis mediated by the ubiquitin-proteasome system. Therefore, the UPS is ideally suited for the remodeling of the embryonic stem cell proteome during the transition from pluripotent to differentiated states and its inverse, the generation of inducible pluripotent stem cells. The Trim-NHL family member LIN41 is among the first E3 ubiquitin ligases to be linked to stem cell pluripotency and reprogramming. Initially discovered in C. elegans as a downstream target of the let-7 miRNA, LIN41 is now recognized as a critical regulator of stem cell fates as well as the timing of neurogenesis. Despite being indispensable for embryonic development and neural tube closure in mice, the underlying mechanisms for LIN41 function in these processes are poorly understood. To better understand the specific contributions of the E3 ligase activity for the stem cell functions of LIN41, we characterized global changes in ubiquitin or ubiquitin-like modifications using Lin41-inducible mouse embryonic stem cells. The tumor suppressor protein p53 was among the five most strongly affected proteins in cells undergoing neural differentiation in response to LIN41 induction. We show that LIN41 interacts with p53, controls its abundance by ubiquitination and antagonizes p53-dependent pro-apoptotic and pro-differentiation responses. In vivo, the lack of LIN41 is associated with upregulation of Grhl3 and widespread caspase-3 activation, two downstream effectors of p53 with essential roles in neural tube closure. As Lin41-deficient mice display neural tube closure defects, we conclude that LIN41 is critical for the regulation of p53 functions in cell fate specification and survival during early brain development.

Immune checkpoint inhibitors have significantly improved the treatment of several cancers. T-cell infiltration and the number of neoantigens caused by tumor-specific mutations are correlated to favorable responses in cancers with a high mutation load. Accordingly, checkpoint immunotherapy is thought to be less effective in tumors with low mutation frequencies such as neuroblastoma, a neuroendocrine tumor of early childhood with poor outcome of the high-risk disease group. However, spontaneous regressions and paraneoplastic syndromes seen in neuroblastoma patients suggest substantial immunogenicity. Using an integrative transcriptomic approach, we investigated the molecular characteristics of T-cell infiltration in primary neuroblastomas as an indicator of pre-existing immune responses and potential responsiveness to checkpoint inhibition. Here, we report that a T-cell-poor microenvironment in primary metastatic neuroblastomas is associated with genomic amplification of the MYCN (N-Myc) proto-oncogene. These tumors exhibited lower interferon pathway activity and chemokine expression in line with reduced immune cell infiltration. Importantly, we identified a global role for N-Myc in the suppression of interferon and pro-inflammatory pathways in human and murine neuroblastoma cell lines. N-Myc depletion potently enhanced targeted interferon pathway activation by a small molecule agonist of the cGAS-STING innate immune pathway. This promoted chemokine expression including Cxcl10 and T-cell recruitment in microfluidics migration assays. Hence, our data suggest N-Myc inhibition plus targeted IFN activation as adjuvant strategy to enforce cytotoxic T-cell recruitment in MYCN -amplified neuroblastomas.

Well-defined gradients of the lipid mediator sphingosine-1-phosphate (S1P) direct chemotactic egress of mature thymocytes from the thymus into the circulation. Although it is known that these gradients result from low S1P levels in the thymic parenchyma and high S1P concentrations at the exit sites and in the plasma, the biochemical mechanisms that regulate these differential S1P levels remain unclear. Several studies demonstrated that ceramide synthase 2 (Cers2) regulates the levels of the S1P precursor sphingosine. We, therefore, investigated whether Cers2 is involved in the regulation of S1P gradients and S1P-dependent egress into the circulation. By analyzing Cers2-deficient mice, we demonstrate that Cers2 limits the levels of S1P in thymus and blood to maintain functional S1P gradients that mediate thymocyte emigration into the circulation. This function is specific for Cers2, as we also show that Cers4 is not involved in the regulation of thymic egress. Our study identified Cers2 as an important regulator of S1P-dependent thymic egress, and thus contributes to the understanding of how S1P gradients are maintained in vivo., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tissue osmolarity varies among different organs and can be considerably increased under pathologic conditions. Hyperosmolarity has been associated with altered stimulatory properties of immune cells, especially macrophages and dendritic cells. We have recently reported that dendritic cells upon exposure to hypertonic stimuli shift their profile towards a macrophage-M2-like phenotype, resulting in attenuated local alloreactivity during acute kidney graft rejection. Here, we examined how hyperosmotic microenvironment affects the cross-priming capacity of dendritic cells. Using ovalbumin as model antigen, we showed that exposure of dendritic cells to hyperosmolarity strongly inhibits activation of antigen-specific T cells despite enhancement of antigen uptake, processing and presentation. We identified TRIF as key mediator of this phenomenon. Moreover, we detected a hyperosmolarity-triggered, TRIF-dependent clustering of MHCI loaded with the ovalbumin-derived epitope, but not of overall MHCI molecules, providing a possible explanation for a reduced T cell activation. Our findings identify dendritic cells as important players in hyperosmolarity-mediated immune imbalance and provide evidence for a novel pathway of inhibition of antigen specific CD8 + T cell response in a hypertonic micromilieu.

Leukocyte trafficking is crucial to facilitate efficient immune responses. Here, we report that the large GTPase dynamin2, which is generally considered to have a key role in endocytosis and membrane remodeling, is an essential regulator of integrin-dependent human T lymphocyte adhesion and migration. Chemical inhibition or knockdown of dynamin2 expression significantly reduced integrin-dependent T cell adhesion in vitro. This phenotype was not observed when T cells were treated with various chemical inhibitors which abrogate endocytosis or actin polymerization. We furthermore detected dynamin2 in signaling complexes and propose that it controls T cell adhesion via FAK/Pyk2- and RapGEF1-mediated Rap1 activation. In addition, the dynamin2 inhibitor-induced reduction of lymphocyte adhesion can be rescued by Rap1a overexpression. We demonstrate that the dynamin2 effect on T cell adhesion does not involve integrin affinity regulation but instead relies on its ability to modulate integrin valency. Taken together, we suggest a previously unidentified role of dynamin2 in the regulation of integrin-mediated lymphocyte adhesion via a Rap1 signaling pathway.

Adaptive cellular immunity is initiated by antigen-specific interactions between T lymphocytes and dendritic cells (DCs). Plasmacytoid DCs (pDCs) support antiviral immunity by linking innate and adaptive immune responses. Here we examined pDC spatiotemporal dynamics during viral infection to uncover when, where, and how they exert their functions. We found that pDCs accumulated at sites of CD8 + T cell antigen-driven activation in a CCR5-dependent fashion. Furthermore, activated CD8 + T cells orchestrated the local recruitment of lymph node-resident XCR1 chemokine receptor-expressing DCs via secretion of the XCL1 chemokine. Functionally, this CD8 + T cell-mediated reorganization of the local DC network allowed for the interaction and cooperation of pDCs and XCR1 + DCs, thereby optimizing XCR1 + DC maturation and cross-presentation. These data support a model in which CD8 + T cells upon activation create their own optimal priming microenvironment by recruiting additional DC subsets to the site of initial antigen recognition., (Published by Elsevier Inc.)

N-glycosylation is generally accepted to enhance the immunogenicity of antigens because of two main reasons. First, the attachment of glycans enables recognition by endocytic receptors like the mannose receptor (MR) and hence increased uptake by dendritic cells (DCs). Second, foreign glycans are postulated to be immunostimulatory and their recognition could induce DC activation. However, a direct comparison between the immunogenicity of N-glycosylated vs. de-glycosylated proteins in vivo and a direct effect of N-glycosylated antigens on the intrinsic capacity of DCs to activate T cells have not been assessed so far.To analyze whether enforced N-glycosylation is a suited strategy to enhance the immunogenicity of non-glycosylated antigens for vaccination studies, we targeted non-glycoproteins towards the MR by introduction of artificial N-glycosylation using the methylotrophic yeast Komagataella phaffii (previously termed Pichia pastoris). We could demonstrate that the introduction of a single N-X-S/T motif was sufficient for efficient MR-binding and internalization. However, addition of N-glycosylated proteins neither influenced DC maturation nor their general capacity to activate T cells, pointing out that enforced N-glycosylation does not increase the immunogenicity of the antigen per se. Additionally, increased antigen-specific cytotoxic T cell responses in vivo after injection of N-glycosylated compared to de-glycosylated proteins were observed but this effect strongly depended on the epitope tested. A beneficial effect of N-glycosylation on antibody production could not be detected, which might be due to MR-cross-linking on DCs and to concomitant differences in IL-6 production by CD4+ T cells.These observations point out that the effect of N-glycosylation on antigen immunogenicity can vary between different antigens and therefore might have important implications for the development of vaccines using K. phaffii.

To determine whether N K cells are involved in the regulation of the antibody response, we studied the effects of human NK clones on B-cell growth and differentiation and the mechanisms involved. We demonstrate that various human NK clones enhance the immunoglobulin production of SAC/rlL-2-activated B cells, e.g. IgG and IgM by up to 23% and anti-tetanus toxoid antibodies by up to430%.Cell cell interactions via cell-surface structures. e.g. the CD11a/ CDI8 molecule, were found lo be critical. Subsequently the NK-mediated B-cell regulation involves cytokines, since cell-free supernatants obtained by 48-h cultures of NK clones exerted BCGF and BCDF activity. Neutralization studies and direct determination characterized these cytokines as IFN-γ and TNF-α. The cytokine production of NK clones could be triggered by activated B cells only. Northern blot analysis demonstrated that activated B cells in co-culture with NK clones were able to induce accumulation of mRNA transcripts for IFN-γ and TNF-α in NK cells. [ABSTRACT FROM AUTHOR]

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality., Competing Interests: The authors declare no conflict of interest.