Cell shape sensing licenses dendritic cells for homeostatic migration to lymph nodes.

Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKß-NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.

Inherited C-terminal TREX1 variants disrupt homology-directed repair to cause senescence and DNA damage phenotypes in Drosophila, mice, and humans.

Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages.

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.

A druggable copper-signalling pathway that drives inflammation.

Inflammation is a complex physiological process triggered in response to harmful stimuli1. It involves cells of the immune system capable of clearing sources of injury and damaged tissues. Excessive inflammation can occur as a result of infection and is a hallmark of several diseases2-4. The molecular bases underlying inflammatory responses are not fully understood. Here we show that the cell surface glycoprotein CD44, which marks the acquisition of distinct cell phenotypes in the context of development, immunity and cancer progression, mediates the uptake of metals including copper. We identify a pool of chemically reactive copper(II) in mitochondria of inflammatory macrophages that catalyses NAD(H) redox cycling by activating hydrogen peroxide. Maintenance of NAD+ enables metabolic and epigenetic programming towards the inflammatory state. Targeting mitochondrial copper(II) with supformin (LCC-12), a rationally designed dimer of metformin, induces a reduction of the NAD(H) pool, leading to metabolic and epigenetic states that oppose macrophage activation. LCC-12 interferes with cell plasticity in other settings and reduces inflammation in mouse models of bacterial and viral infections. Our work highlights the central role of copper as a regulator of cell plasticity and unveils a therapeutic strategy based on metabolic reprogramming and the control of epigenetic cell states.

Primary role of type I interferons for the induction of functionally optimal antigen-specific CD8+ T cells in HIV infection.

BACKGROUND: CD8+ T cells equipped with a full arsenal of antiviral effector functions are critical for effective immune control of HIV-1. It has nonetheless remained unclear how best to elicit such potent cellular immune responses in the context of immunotherapy or vaccination. HIV-2 has been associated with milder disease manifestations and more commonly elicits functionally replete virus-specific CD8+ T cell responses compared with HIV-1. We aimed to learn from this immunological dichotomy and to develop informed strategies that could enhance the induction of robust CD8+ T cell responses against HIV-1. METHODS: We developed an unbiased in vitro system to compare the de novo induction of antigen-specific CD8+ T cell responses after exposure to HIV-1 or HIV-2. The functional properties of primed CD8+ T cells were assessed using flow cytometry and molecular analyses of gene transcription. FINDINGS: HIV-2 primed functionally optimal antigen-specific CD8+ T cells with enhanced survival properties more effectively than HIV-1. This superior induction process was dependent on type I interferons (IFNs) and could be mimicked via the adjuvant delivery of cyclic GMP-AMP (cGAMP), a known agonist of the stimulator of interferon genes (STING). CD8+ T cells elicited in the presence of cGAMP were polyfunctional and highly sensitive to antigen stimulation, even after priming from people living with HIV-1. INTERPRETATION: HIV-2 primes CD8+ T cells with potent antiviral functionality by activating the cyclic GMP-AMP synthase (cGAS)/STING pathway, which results in the production of type I IFNs. This process may be amenable to therapeutic development via the use of cGAMP or other STING agonists to bolster CD8+ T cell-mediated immunity against HIV-1. FUNDING: This work was funded by INSERM, the Institut Curie, and the University of Bordeaux (Senior IdEx Chair) and by grants from Sidaction (17-1-AAE-11097, 17-1-FJC-11199, VIH2016126002, 20-2-AEQ-12822-2, and 22-2-AEQ-13411), the Agence Nationale de la Recherche sur le SIDA (ECTZ36691, ECTZ25472, ECTZ71745, and ECTZ118797), and the Fondation pour la Recherche Médicale (EQ U202103012774). D.A.P. was supported by a Wellcome Trust Senior Investigator Award (100326/Z/12/Z).

RELA tunes innate-like interferon I/III responses in human T cells.

In innate immune cells, intracellular sensors such as cGAS-STING stimulate type I/III interferon (IFN) expression, which promotes antiviral defense and immune activation. However, how IFN-I/III expression is controlled in adaptive cells is poorly understood. Here, we identify a transcriptional rheostat orchestrated by RELA that confers human T cells with innate-like abilities to produce IFN-I/III. Despite intact cGAS-STING signaling, IFN-I/III responses are stunted in CD4+ T cells compared with dendritic cells or macrophages. We find that lysine residues in RELA tune the IFN-I/III response at baseline and in response to STING stimulation in CD4+ T cells. This response requires positive feedback driven by cGAS and IRF7 expression. By combining RELA with IRF3 and DNA demethylation, IFN-I/III production in CD4+ T cells reaches levels observed in dendritic cells. IFN-I/III production provides self-protection of CD4+ T cells against HIV infection and enhances the elimination of tumor cells by CAR T cells. Therefore, innate-like functions can be tuned and leveraged in human T cells.

Selective STING stimulation in dendritic cells primes antitumor T cell responses.

T cells that recognize tumor antigens are crucial for mounting antitumor immune responses. Induction of antitumor T cells in immunogenic tumors depends on STING, the intracellular innate immune receptor for cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and related cyclic dinucleotides (CDNs). However, the optimal way to leverage STING activation in nonimmunogenic tumors is still unclear. Here, we show that cGAMP delivery by intratumoral injection of virus-like particles (cGAMP-VLP) led to differentiation of circulating tumor-specific T cells, decreased tumor regulatory T cells (Tregs), and antitumoral responses that synergized with PD1 blockade. By contrast, intratumoral injection of the synthetic CDN ADU-S100 led to tumor necrosis and systemic T cell activation but simultaneously depleted immune cells from injected tumors and induced minimal priming of circulating tumor-specific T cells. The antitumor effects of cGAMP-VLP required type 1 conventional dendritic cells (cDC1), whereas ADU-S100 eliminated cDC1 from injected tumors. cGAMP-VLP preferentially targeted STING in dendritic cells at a 1000-fold smaller dose than ADU-S100. Subcutaneous administration of cGAMP-VLP showed synergy when combined with PD1 blockade or a tumor Treg-depleting antibody to elicit systemic tumor-specific T cells and antitumor activity, leading to complete and durable tumor eradication in the case of tumor Treg depletion. These findings show that cell targeting of STING stimulation shapes the antitumor T cell response and identify a therapeutic strategy to enhance T cell-targeted immunotherapy.

Deficiency for SAMHD1 activates MDA5 in a cGAS/STING-dependent manner.

Defects in nucleic acid metabolizing enzymes can lead to spontaneous but selective activation of either cGAS/STING or RIG-like receptor (RLR) signaling, causing type I interferon-driven inflammatory diseases. In these pathophysiological conditions, activation of the DNA sensor cGAS and IFN production are linked to spontaneous DNA damage. Physiological, or tonic, IFN signaling on the other hand is essential to functionally prime nucleic acid sensing pathways. Here, we show that low-level chronic DNA damage in mice lacking the Aicardi-Goutières syndrome gene SAMHD1 reduced tumor-free survival when crossed to a p53-deficient, but not to a DNA mismatch repair-deficient background. Increased DNA damage did not result in higher levels of type I interferon. Instead, we found that the chronic interferon response in SAMHD1-deficient mice was driven by the MDA5/MAVS pathway but required functional priming through the cGAS/STING pathway. Our work positions cGAS/STING upstream of tonic IFN signaling in Samhd1-deficient mice and highlights an important role of the pathway in physiological and pathophysiological innate immune priming.

Extracellular vesicles from triple negative breast cancer promote pro-inflammatory macrophages associated with better clinical outcome.

Tumor associated macrophages (TAMs), which differentiate from circulating monocytes, are pervasive across human cancers and comprise heterogeneous populations. The contribution of tumor-derived signals to TAM heterogeneity is not well understood. In particular, tumors release both soluble factors and extracellular vesicles (EVs), whose respective impact on TAM precursors may be different. Here, we show that triple negative breast cancer cells (TNBCs) release EVs and soluble molecules promoting monocyte differentiation toward distinct macrophage fates. EVs specifically promoted proinflammatory macrophages bearing an interferon response signature. The combination in TNBC EVs of surface CSF-1 promoting survival and cargoes promoting cGAS/STING or other activation pathways led to differentiation of this particular macrophage subset. Notably, macrophages expressing the EV-induced signature were found among patients' TAMs. Furthermore, higher expression of this signature was associated with T cell infiltration and extended patient survival. Together, this data indicates that TNBC-released CSF-1-bearing EVs promote a tumor immune microenvironment associated with a better prognosis in TNBC patients.

Virus-stimulated Dendritic Cells Elicit a T Antiviral Transcriptional Signature in Human CD4+ Lymphocytes.

Dendritic cells (DCs) play a pivotal role in the functional differentiation of CD4+ T cells in response to pathogens. In CD4+ T cells, HIV-1 replicates efficiently, while HIV-2, a related virus of reduced pathogenicity, is better controlled. How the DC response to HIV-1 vs HIV-2 contributes to programming an antiviral state in CD4+ T cells is not known. Here, we identify a transcriptional signature associated with progressive resistance to HIV infection in CD4+ T cells. We developed a model of naïve CD4+ T cell priming by DCs stimulated with a panel of seven viruses or synthetic ligands for the viral nucleic acid sensors cGAS and TLRs. DCs produced a cytokine response to HIV-2 infection more similar to the response to cGAS ligands than TLR ligands. In response to these signals, naive CD4+ T cells acquired a gradual antiviral resistance to subsequent HIV infection. The antiviral state was concomitant with the induction of the TH1 cytokine IFNγ and the type I interferon-stimulated gene (ISG) MX1, while the TFH cytokine IL-21 was not increased. By performing a transcriptional network analysis in T cells, we identified five distinct gene modules with characteristic ISG, TH1, TFH, IFN-I and proliferative signatures. Finally, we leverage this module to assemble a T antiviral signature of 404 genes that correlate with the antiviral state in T cells. Altogether, the study illuminates the programming of the antiviral state in T cells. The T antiviral gene signature in human CD4+ lymphocytes constitutes a resource for genetic screens and genomics analysis.

Discovery of Genes that Modulate Flavivirus Replication in an Interferon-Dependent Manner.

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.

Inhibition of HIV infection by structural proteins of the inner nuclear membrane is associated with reduced chromatin dynamics.

The human immunodeficiency virus (HIV) enters the nucleus to establish infection, but the role of nuclear envelope proteins in this process is incompletely understood. Inner nuclear transmembrane proteins SUN1 and SUN2 connect nuclear lamins to the cytoskeleton and participate in the DNA damage response (DDR). Increased levels of SUN1 or SUN2 potently restrict HIV infection through an unresolved mechanism. Here, we find that the antiviral activities of SUN1 and SUN2 are distinct. HIV-1 and HIV-2 are preferentially inhibited by SUN1 and SUN2, respectively. We identify DNA damage inducers that stimulate HIV-1 infection and show that SUN1, but not SUN2, neutralizes this effect. Finally, we show that chromatin movements and nuclear rotations are associated with the effects of SUN proteins and Lamin A/C on infection. These results reveal an emerging role of chromatin dynamics and the DDR in the control of HIV infection by structural components of the nuclear envelope.

Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Single-cell analysis reveals divergent responses of human dendritic cells to the MVA vaccine.

Modified vaccinia Ankara (MVA) is a live, attenuated human smallpox vaccine and a vector for the development of new vaccines against infectious diseases and cancer. Efficient activation of the immune system by MVA partially relies on its encounter with dendritic cells (DCs). MVA infection of DCs leads to multiple outcomes, including cytokine production, activation of costimulatory molecules for T cell stimulation, and cell death. Here, we examined how these diverse responses are orchestrated in human DCs. Single-cell analyses revealed that the response to MVA infection in DCs was limited to early viral gene expression. In response to the early events in the viral cycle, we found that DCs grouped into three distinct clusters. A cluster of infected cells sensed the MVA genome by the intracellular innate immunity pathway mediated by cGAS, STING, TBK1, and IRF3 and subsequently produced inflammatory cytokines. In response to these cytokines, a cluster of noninfected bystander cells increased costimulatory molecule expression. A separate cluster of infected cells underwent caspase-dependent apoptosis. Induction of apoptosis persisted after inhibition of innate immunity pathway mediators independently of previously described IRF-dependent or replication-dependent pathways and was a response to early MVA gene expression. Together, our study identified multiple mechanisms that underlie the interactions of MVA with human DCs.

A genetic memory initiates the epigenetic loop necessary to preserve centromere position.

Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-AOFF/ON ). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4+ T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.

Mutations in COPA lead to abnormal trafficking of STING to the Golgi and interferon signaling.

Heterozygous missense mutations in coatomer protein subunit α, COPA, cause a syndrome overlapping clinically with type I IFN-mediated disease due to gain-of-function in STING, a key adaptor of IFN signaling. Recently, increased levels of IFN-stimulated genes (ISGs) were described in COPA syndrome. However, the link between COPA mutations and IFN signaling is unknown. We observed elevated levels of ISGs and IFN-α in blood of symptomatic COPA patients. In vitro, both overexpression of mutant COPA and silencing of COPA induced STING-dependent IFN signaling. We detected an interaction between COPA and STING, and mutant COPA was associated with an accumulation of ER-resident STING at the Golgi. Given the known role of the coatomer protein complex I, we speculate that loss of COPA function leads to enhanced type I IFN signaling due to a failure of Golgi-to-ER STING retrieval. These data highlight the importance of the ER-Golgi axis in the control of autoinflammation and inform therapeutic strategies in COPA syndrome.

Immunometabolism pathways as the basis for innovative anti-viral strategies (INITIATE): A Marie Sklodowska-Curie innovative training network.

The past century has witnessed major advances in the control of many infectious diseases, yet outbreaks and epidemics caused by (re-) emerging RNA viruses continue to pose a global threat to human health. As illustrated by the global COVID19 pandemic, high healthcare costs, economic disruption and loss of productivity reinforce the unmet medical need to develop new antiviral strategies to combat not only the current pandemic but also future viral outbreaks. Pivotal for effective anti-viral defense is the innate immune system, a first line host response that senses and responds to virus infection. While molecular details of the innate immune response are well characterized, this research field is now being revolutionized with the recognition that cell metabolism has a major impact on the antiviral and inflammatory responses to virus infections. A detailed understanding of the role of metabolic regulation with respect to antiviral and inflammatory responses, together with knowledge of the strategies used by viruses to exploit immunometabolic pathways, will ultimately change our understanding and treatment of pathogenic viral diseases. INITIATE is a Marie Sklodowska-Curie Actions Innovative Training Network (MSCA-ITN), with the goal to train 15 early stage PhD researchers (ESRs) to become experts in antiviral immunometabolism (https://initiate-itn.eu/). To this end, INITIATE brings together a highly complementary international team of academic and corporate leaders from 7 European countries, with outstanding track records in the historically distinct research fields of virology, immunology and metabolism. The ESRs of INITIATE are trained in these interdisciplinary research fields through individual investigator-driven research projects, specialized scientific training events, workshops on academia-industry interactions, outreach & communication. INITIATE will deliver a new generation of creative and entrepreneurial researchers who will be able to face the inevitable future challenges in combating viral diseases.

A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway.

A subset of TLRs is specialised in the detection of incoming pathogens by sampling endosomes for nucleic acid contents. Among them, TLR3 senses the abnormal presence of double-stranded RNA in the endosomes and initiates a potent innate immune response via activation of NF-κB and IRF3. Nevertheless, mechanisms governing TLR3 regulation remain poorly defined. To identify new molecular players involved in the TLR3 pathway, we performed a genome-wide screen using CRISPR/Cas9 technology. We generated TLR3+ reporter cells carrying a NF-κB-responsive promoter that controls GFP expression. Cells were next transduced with a single-guide RNA (sgRNA) library, subjected to sequential rounds of stimulation with poly(I:C) and sorting of the GFP-negative cells. Enrichments in sgRNA estimated by deep sequencing identified genes required for TLR3-induced activation of NF-κB. Among the hits, five genes known to be critically involved in the TLR3 pathway, including TLR3 itself and the chaperone UNC93B1, were identified by the screen, thus validating our strategy. We further studied the top 40 hits and focused on the transcription factor aryl hydrocarbon receptor (AhR). Depletion of AhR had a dual effect on the TLR3 response, abrogating IL-8 production and enhancing IP-10 release. Moreover, in primary human macrophages exposed to poly(I:C), AhR activation enhanced IL-8 and diminished IP-10 release. Overall, these results reveal AhR plays a role in the TLR3 cellular innate immune response.

A Comprehensive Map of the Monocyte-Derived Dendritic Cell Transcriptional Network Engaged upon Innate Sensing of HIV.

Transcriptional programming of the innate immune response is pivotal for host protection. However, the transcriptional mechanisms that link pathogen sensing with innate activation remain poorly understood. During HIV-1 infection, human dendritic cells (DCs) can detect the virus through an innate sensing pathway, leading to antiviral interferon and DC maturation. Here, we develop an iterative experimental and computational approach to map the HIV-1 innate response circuitry in monocyte-derived DCs (MDDCs). By integrating genome-wide chromatin accessibility with expression kinetics, we infer a gene regulatory network that links 542 transcription factors with 21,862 target genes. We observe that an interferon response is required, yet insufficient, to drive MDDC maturation and identify PRDM1 and RARA as essential regulators of the interferon response and MDDC maturation, respectively. Our work provides a resource for interrogation of regulators of HIV replication and innate immunity, highlighting complexity and cooperativity in the regulatory circuit controlling the response to infection.