Jun dependent mTAM differentiation confirmed by high-dimensional spectral flow cytometry.

Analysis of equivalent TAM states and JUN activity in human melanoma.

Additional analysis of scRNA-seq analysis of TAMs from YUMM1.7 tumors.

Immune-phenotyping YUMM1.7 tumours from JunΔCsf1r mice.

Abstract Tumor-associated macrophages (TAMs) play important roles in cancer progression and resistance to therapy. Recent studies have shown that TAMs include both long-lived resident tissue macrophages (RTMs) and short-lived monocyte-derived macrophages (MDMs) with limited proliferative potential. RTMs and MDMs have been suggested to play divergent roles in tumorigenesis; RTMs are aligned with trophic functions, whereas MDMs are enriched for immune-regulatory pathways. Here we established a specific role for the AP-1 factor JUN in the differentiation and maintenance of MDMs and the specification of pro-tumoral trophic functions during tumor development. Alternatively, the immune-regulatory functions of TAMs remained JUN-independent. JUN was required for the specification and maintenance of pro-tumoral TAMs that support blood vessel maturation and tumor growth. Single-cell transcriptomics analysis uncovered the alternative fates for tumor-infiltrating monocytes and the development of distinct TAM states associated with trophic functions and immune-regulation. These studies demonstrate an important role for JUN in the specification of pro-tumoral monocyte-derived TAMs that could offer opportunities for selective TAM-targeted therapies for cancer. Significance: JUN promotes the specification of pro-tumoral macrophages but is dispensable for pro-inflammatory macrophages, suggesting targeting the JUN pathway in myeloid cells could suppress pro-tumoral macrophages while sparing antitumor inflammatory myeloid cells.

Defective angiogenesis in JunΔCsf1r mice and rescue with recombinant sFLT1.

Characterization of monosyte-derived TAMs in YUMM1.7 melanomas.

CSF1 blockade in zymosan peritonitis.

Characterization of Jun deletion JunΔCsf1r mice.

Characterization of Jun deficient macrophages in vitro.

The small chloroplastic protein CP12 has multiple functions, including the regulation of enzymes in the Calvin-Benson-Bassham cycle. Here, we investigated its role in the acclimation of Chlamydomonas reinhardtii to varying CO2 availability. This alga has a CO2 concentrating mechanism that increases the supply of CO2 to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and involves hallmarks such as HCO3- transporters and carbonic anhydrases as well as the condensation of RuBisCO within the pyrenoid via its interaction with a scaffold protein named Essential Pyrenoid Component 1 (EPYC1). We showed that compared to the wild type, at high CO2, C. reinhardtii CP12 deletion mutants, or partially complemented mutants, have less phosphoribulokinase and ribulose-1,5-bisphosphate (RuBP) indicating that the regeneration of RuBP is regulated by CP12. In the absence of CP12, the expected relocation of RuBisCO towards the pyrenoid was not observed upon transition from high to very low CO2, contrary to WT cells. The CP12 deletion mutants are a unique example where the induction of CO2 concentrating mechanism hallmarks at very low CO2 was not accompanied by RuBisCO relocation. Altogether, these results suggest that CP12 contributes to the coordination between RuBP regeneration, RuBisCO location and CO2 acquisition. Highlight CP12 regulates phosphoribulokinase amount and its product RuBP. The CP12 deletion mutants are unique example where the RuBisCO location was not correlated with the induction of CO2 concentration mechanism. This reveals a novel link between Calvin-Benson-Bassham cycle regulation and CO2 concentrating mechanisms in Chlamydomonas reinhardtii . ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

A. baumannii can rapidly acquire new resistance mechanisms and persist on abiotic surface, enabling the colonization of asymptomatic human host. In Acinetobacter the type VI secretion system (T6SS) is involved in twitching, surface motility and is used for interbacterial competition allowing the bacteria to uptake DNA. A. baumannii possesses a T6SS that has been well studied for its regulation and specific activity, but little is known concerning its assembly and architecture. The T6SS nanomachine is built from three architectural sub-complexes. Unlike the baseplate (BP) and the tail-tube complex (TTC), which are inherited from bacteriophages, the membrane complex (MC) originates from bacteria. The MC is the most external part of the T6SS and, as such, is subjected to evolution and adaptation. One unanswered question on the MC is how such a gigantesque molecular edifice is inserted and crosses the bacterial cell envelope. The A. baumannii MC lacks an essential component, the TssJ lipoprotein, which anchors the MC to the outer membrane. In this work, we studied how A. baumannii compensates the absence of a TssJ. We have characterized for the first time the A. baumannii's specific T6SS MC, its unique characteristic, its membrane localization, and assembly dynamics. We also defined its composition, demonstrating that its biogenesis employs three Acinetobacter-specific envelope-associated proteins that define an intricate network leading to the assembly of a five-proteins membrane super-complex. Our data suggest that A. baumannii has divided the function of TssJ by (1) co-opting a new protein TsmK that stabilizes the MC and by (2) evolving a new domain in TssM for homo-oligomerization, a prerequisite to build the T6SS channel. We believe that the atypical species-specific features we report in this study will have profound implication in our understanding of the assembly and evolutionary diversity of different T6SSs, that warrants future investigation.

Conventional type 1 dendritic cells (cDC1s) are critical for antitumor immunity. They acquire antigens from dying tumor cells and cross-present them to CD8+ T cells, promoting the expansion of tumor-specific cytotoxic T cells. However, the signaling pathways that govern the antitumor functions of cDC1s in immunogenic tumors are poorly understood. Using single-cell transcriptomics to examine the molecular pathways regulating intratumoral cDC1 maturation, we found nuclear factor κB (NF-κB) and interferon (IFN) pathways to be highly enriched in a subset of functionally mature cDC1s. We identified an NF-κB-dependent and IFN-γ-regulated gene network in cDC1s, including cytokines and chemokines specialized in the recruitment and activation of cytotoxic T cells. By mapping the trajectory of intratumoral cDC1 maturation, we demonstrated the dynamic reprogramming of tumor-infiltrating cDC1s by NF-κB and IFN signaling pathways. This maturation process was perturbed by specific inactivation of either NF-κB or IFN regulatory factor 1 (IRF1) in cDC1s, resulting in impaired expression of IFN-γ-responsive genes and consequently a failure to efficiently recruit and activate antitumoral CD8+ T cells. Last, we demonstrate the relevance of these findings to patients with melanoma, showing that activation of the NF-κB/IRF1 axis in association with cDC1s is linked with improved clinical outcome. The NF-κB/IRF1 axis in cDC1s may therefore represent an important focal point for the development of new diagnostic and therapeutic approaches to improve cancer immunotherapy.

Macrophages possess intrinsic tumoricidal activity, yet tumor-associated macrophages (TAMs) rapidly adopt an alternative phenotype within the tumor microenvironment that is marked by tumor-promoting immunosuppressive and trophic functions. The mechanisms that promote such TAM polarization remain poorly understood, but once identified, they may represent important therapeutic targets to block the tumor-promoting functions of TAMs and restore their anti-tumor potential. Here, we have characterized TAMs in a mouse model of metastatic ovarian cancer. We show that ovarian cancer cells promote membrane-cholesterol efflux and depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4-mediated reprogramming, including inhibition of IFNγ-induced gene expression. Genetic deletion of ABC transporters, which mediate cholesterol efflux, reverts the tumor-promoting functions of TAMs and reduces tumor progression. These studies reveal an unexpected role for membrane-cholesterol efflux in driving TAM-mediated tumor progression while pointing to a potentially novel anti-tumor therapeutic strategy.

The spleen plays an important role in protective immunity to bloodborne pathogens. Macrophages and dendritic cells (DCs) in the spleen marginal zone capture microbial antigens to trigger adaptive immune responses. Marginal zone macrophages (MZMs) can also act as a replicative niche for intracellular pathogens, providing a platform for mounting the immune response. Here, we describe a role for RANK in the coordinated function of antigen-presenting cells in the spleen marginal zone and triggering anti-viral immunity. Targeted deletion of RANK results in the selective loss of CD169+ MZMs, which provide a niche for viral replication, while RANK signaling in DCs promotes the recruitment and activation of anti-viral memory CD8 T cells. These studies reveal a role for the RANKL/RANK signaling axis in the orchestration of protective immune responses in the spleen marginal zone that has important implications for the host response to viral infection and induction of acquired immunity.

Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKβ in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity.

The Notch signalling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase, conserved from Drosophila to humans, which has been proposed to be a regulator of Notch signalling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal zone B cell commitment. Deltex-1 was also found to be highly expressed in germinal center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1 mice were viable and fertile. A detailed histological analysis did not reveal any defect in major organs. Tand B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular from for a fourth deltex gene identified during the course of this study are discussed. 3 ha l-0 01 65 74 1, v er si on 1 11 S ep 2 00 8

BACKGROUND:The INK4/ARF locus encodes three tumor suppressor genes (p15(Ink4b), Arf and p16(Ink4a)) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized.PRINCIPAL FINDINGS:Here we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the INK4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the INK4a/ARF locus during senescence.CONCLUSIONS:We identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the INK4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the INK4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the INK4/ARF locus.

The TCRβ gene enhancer (Eβ) commands TCRβ gene expression through the lifespan of T lymphocytes. Genetic and molecular studies have implied that in early thymocytes, Eβ directs chromatin opening over the Dβ-Jβ-Cβ domains and triggers initial Dβ-Jβ recombination. In mature T cells, Eβ is required for expression of the assembled TCRβ gene. Whether these separate activities rely on distinct Eβ regulatory sequences and involve differing modes of activation is unclear. Using gene targeting in mouse embryonic stem cells, we replaced Eβ by a conserved core fragment (Eβ169). We found that Eβ169-carrying alleles were capable of sustaining β gene expression and the development of mature T cells in homozygous knockin mice. Surprisingly, these procedures and underlying molecular transactions were affected to a wide range of degrees depending on the developmental stage. Early thymocytes barely achieved Dβ-Jβ germline transcription and recombination. In contrast, T cells displayed substantial though heterogeneous levels of VDJ-rearranged TCRβ gene expression. Our results have implications regarding enhancer function in cells of the adaptive immune system and, potentially, TCRβ gene recombination and allelic exclusion.

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actinand Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of abT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca influx and phosphorylation and degradation of the inhibitor of NF-kB (IkB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts abT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages. Citation: Mugnier B, Nal B, Verthuy C, Boyer C, Lam D, et al. (2008) Coronin-1A Links Cytoskeleton Dynamics to TCRab-Induced Cell Signaling. PLoS ONE 3(10): e3467. doi:10.1371/journal.pone.0003467 Editor: Jean Kanellopoulos, University Paris Sud, France Received July 17, 2008; Accepted September 20, 2008; Published October 21, 2008 Copyright: 2008 Mugnier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Inserm, CNRS, the ‘Association pour la Recherche sur le Cancer’ (ARC), the ‘Fondation Princesse Grace de Monaco’, and the Commission of the European Communities (to PF); and from the ‘Ministère de l’Education Nationale et de la Recherche’ (ACI #108) (to PF and AA). BN was supported by fellowships from the ‘Ligue Nationale Contre le Cancer’ and ARC. BM was supported by a fellowship from ARC. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ferrier@ciml.univ-mrs.fr . These authors contributed equally to this work.