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Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that express an invariant T cell receptor α chain and contribute to bridging innate and acquired immunity with rapid production of large amounts of cytokines after stimulation. Among effecter subsets of iNKT cells, follicular helper NKT (NKTFH) cells are specialized to help B cells. However, the mechanisms of NKTFH cell differentiation remain to be elucidated. In this report, we studied the mechanism of NKTFH cell differentiation induced by pneumococcal surface protein A and α-galactosylceramide (P/A) vaccination. We found that Gr-1+ cells helped iNKT cell proliferation and NKTFH cell differentiation in the spleen by producing interleukin-27 (IL-27) in the early phase after vaccination. The neutralization of IL-27 impaired NKTFH cell differentiation, which resulted in compromised antibody production and diminished protection against Streptococcus pneumoniae infection by the P/A vaccine. Our data indicated that Gr-1+ cell-derived IL-27 stimulated mitochondrial metabolism, meeting the energic demand required for iNKT cells to differentiate into NKTFH cells. Interestingly, Gr-1+ cell-derived IL-27 was induced by iNKT cells via interferon-γ production. Collectively, our findings suggest that optimizing the metabolism of iNKT cells was essential for acquiring specific effector functions, and they provide beneficial knowledge on iNKT cell-mediated vaccination-mediated therapeutic strategies. [ABSTRACT FROM AUTHOR]

To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h poststimulation) and disaggregate (2 h poststimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling, whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. In response to lipopolysaccharide treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reorganizes the nuclear landscape to enable inflammatory gene expression following innate stimuli. [ABSTRACT FROM AUTHOR]

Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer. [ABSTRACT FROM AUTHOR]

Copper is an essential trace element for the human body, and its requirement for optimistic immune functions has been recognized for decades. How copper is involved in the innate immune pathway, however, remains to be clarified. Here, we report that copper serves as a signal molecule to regulate the kinase activity of alpha-kinase 1 (ALPK1), a cytosolic pattern-recognition receptor (PRR), and therefore promotes host cell defense against bacterial infection. We show that in response to infection, host cells actively accumulate copper in the cytosol, and the accumulated cytosolic copper enhances host cell defense against evading pathogens, including intracellular and, unexpectedly, extracellular bacteria. Subsequently, we demonstrate that copper activates the innate immune pathway of host cells in an ALPK1-dependent manner. Further mechanistic studies reveal that copper binds to ALPK1 directly and is essential for the kinase activity of this cytosolic PRR. Moreover, the binding of copper to ALPK1 enhances the sensitivity of ALPK1 to the bacterial metabolite ADP-heptose and eventually prompts host cells to elicit an enhanced immune response during bacterial infection. Finally, using a zebrafish in vivo model, we show that a copper-treated host shows an increased production of proinflammatory cytokines, enhanced recruitment of phagosome cells, and promoted bacterial clearance. Our findings uncover a previously unrecognized role of copper in the modulation of host innate immune response against bacterial pathogens and advance our knowledge on the cross talk between cytosolic copper homeostasis and immune system. [ABSTRACT FROM AUTHOR]

The enzyme cyclic GMP-AMP synthase (cGAS) is a key sensor for detecting misplaced double-stranded DNA (dsDNA) of genomic, mitochondrial, and microbial origin. It synthesizes 2'3'-cGAMP, which in turn activates the stimulator of interferon genes pathway, leading to the initiation of innate immune responses. Here, we identified Listerin as a negative regulator of cGAS-mediated innate immune response. We found that Listerin interacts with cGAS on endosomes and promotes its K63-linked ubiquitination through recruitment of the E3 ligase TRIM27. The polyubiquitinated cGAS is then recognized by the endosomal sorting complexes required for transport machinery and sorted into endosomes for degradation. Listerin deficiency enhances the innate antiviral response to herpes simplex virus 1 infection. Genetic deletion of Listerin also deteriorates the neuroinflammation and the ALS disease progress in an ALS mice model; overexpression of Listerin can robustly ameliorate disease progression in ALS mice. Thus, our work uncovers a mechanism for cGAS regulation and suggests that Listerin may be a promising therapeutic target for ALS disease. [ABSTRACT FROM AUTHOR]

Autoantibodies directed against complement component C1q are commonly associated with autoimmune diseases, especially systemic lupus erythematosus. Importantly, these anti-C1q autoantibodies are specific for ligand-bound, solid-phase C1q and do not bind to fluid-phase C1q. In patients with anti-C1q, C1q levels are in the normal range, and the autoantibodies are thus not depleting. To study these human anti-C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and recombinantly produced nine monoclonal antibodies (mAbs) from four different healthy individuals. The isolated mAbs were of the IgG isotype, contained extensively mutated variable domains, and showed high affinity to the collagen-like region of C1q. The anti-C1q mAbs exclusively bound solid-phase C1q in complex with its natural ligands, including immobilized or antigen-bound IgG, IgM or CRP, and necrotic cells. Competition experiments reveal that at least 2 epitopes, also targeted by anti-C1q antibodies in sera from SLE patients, are recognized. Electron microscopy with hexameric IgG-C1q immune complexes demonstrated that multiple mAbs can interact with a single C1q molecule and identified the region of C1q targeted by these mAbs. The opsonization of immune complexes with anti-C1q greatly enhanced Fc-receptor-mediated phagocytosis but did not increase complement activation. We conclude that human anti-C1q autoantibodies specifically bind neo-epitopes on solid-phase C1q, which results in an increase in Fc-receptor-mediated effector functions that may potentially contribute to autoimmune disease immunopathology. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGF-β) directly acts on naive, effector, and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-β signaling. TGF-β availability is altered by infections and cancer; however, the dose-dependent effects of TGF-β on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-β signals interact to shape the functional outcome of Tmem reactivation. We found that TGF-β could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or proinflammatory signals. In contrast, even high doses of TGF-β had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 Tmem may not always receive TGF-β signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-β signals is of importance. We found that exposure to TGF-β before or after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-β altered ~10% of the regulatory elements induced by reactivation and also elicited transcriptional changes indicative of broadly modulated functional properties. We confirmed some changes on the protein level and found that TGF-β-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-β is not simply suppressing CD8 Tmem but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

In multiple sclerosis (MS), pathogenic T cell responses are known to be important drivers of autoimmune inflammation. However, increasing evidence suggests an additional role for B cells, which may contribute to pathogenesis via antigen presentation and production of proinflammatory cytokines. However, these B cell effector functions are not featured well in classical experimental autoimmune encephalomyelitis (EAE) mouse models. Here, we compared properties of myelin oligodendrocyte glycoprotein (MOG)-specific and polyclonal B cells and developed an adjuvant-free cotransfer EAE mouse model, where highly activated, MOG-specific induced germinal center B cells provide the critical stimulus for disease development. We could show that high levels of MOG-specific immunoglobulin G (IgGs) are not required for EAE development, suggesting that antigen presentation and activation of cognate T cells by B cells may be important for pathogenesis. As our model allows for B cell manipulation prior to transfer, we found that overexpression of the proinflammatory cytokine interleukin (IL)-6 by MOG-specific B cells leads to an accelerated EAE onset accompanied by activation/expansion of the myeloid compartment rather than a changed T cell response. Accordingly, knocking out IL-6 or tumor necrosis factor a in MOG-specific B cells via CRISPR-Cas9 did not affect activation of pathogenic T cells. In summary, we generated a tool to dissect pathogenic B cell effector function in EAE development, which should improve our understanding of pathogenic processes in MS. [ABSTRACT FROM AUTHOR]

Antibody responses against highly conserved epitopes on the stalk domain of influenza virus hemagglutinin (HA) confer broad protection; however, such responses are limited. To effectively induce stalk-specific immunity against conserved HA epitopes, sequential immunization strategies have been developed based on chimeric HA (cHA) constructs featuring different head domains but the same stalk regions. Immunogenicity studies in small animal models, as well as in humans, revealed that cHA immunogens elicit stalk-specific IgG responses with broad specificity against heterologous influenza virus strains. However, the mechanisms by which these antibodies confer in vivo protection and the contribution of their Fc effector function remain unclear. To characterize the role of Fc-FcγR (Fcγ receptor) interactions to the in vivo protective activity of IgG antibodies elicited in participants in a phase I trial of a cHA vaccine candidate, we performed passive transfer studies of vaccine-elicited IgG antibodies in mice humanized for all classes of FcγRs, as well as in mice deficient for FcγRs. IgG antibodies elicited upon cHA vaccination completely protected FcγR humanized mice against lethal influenza virus challenge, while no protection was evident in FcγR-deficient mice, suggesting a major role for FcγR pathways in the protective function of vaccine-elicited IgG antibodies. These findings have important implications for influenza vaccine development, guiding the design of vaccination approaches with the capacity to elicit IgG responses with optimal Fc effector function. [ABSTRACT FROM AUTHOR]

The ability of immune cells to directly interact with transformed cells is an essential component of immune surveillance and critical for optimal tissue function. The tumor-immune interactome (the collective cellular interactions between oncogenic cells and immune cells) is distinct and varied based on the tissue location and immunogenicity of tumor subtypes. However, comprehensive landscape and the consequences of tumor-interacting immune cells in the tumor microenvironment are not well understood. Current tools are limited in their ability to identify and record interactors in vivo or be utilized for downstream analysis. Here, we describe the development and validation of a technology leveraging synthetic Notch receptors reporting physical tumor cell-immune cell contact in vivo in order to decipher the tumor-immune interactome. We call this approach, Tumor-Immune Interactome Non-biased Discovery Retroviral Reporter or TIINDRR. Using TIINDRR, we identify the tumor-immune interactomes that define immunological refractory and sensitive tumors and how different immunotherapies alter these interactions. Thus, TIINDRR provides a flexible and versatile tool for studying in-vivo tumor-immune cell interactions, aiding in the identification of biologically relevant information needed for the rational design of immune-based therapies. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Advanced PDAC is considered incurable. Nearly 90% of pancreatic cancers are caused by oncogenic KRAS mutations. The mechanisms of primary or acquired resistance to KRAS inhibition are currently unknown. Here, we propose that oncogenic dependency, rather than KRAS mutation per se, plays a dominant role in the immune response to cancer, including late-stage PDAC. Classifying tumor samples according to KRAS activity scores allows accurate prediction of tumor immune composition and therapy response. Dual RAS/MAPK pathway blockade combining KRAS and MEK inhibitors is more effective than the selective KRAS inhibitor alone in attenuating MAPK activation and unblocking the influx of T cells into the tumor. Lowering KRAS activity in established tumors promotes immune infiltration, but with a limited antitumor effect, whereas combining KRAS/MEK inhibition with immune checkpoint blockade achieves durable regression in preclinical models. The results are directly applicable to stratifying human PDAC based on KRAS dependency values and immune cell composition to improve therapeutic design. [ABSTRACT FROM AUTHOR]

Variation in human immune response to the same bacterial or viral pathogen is well established in the literature. Variation in immune response to microbial challenge has also been observed within the human oral cavity. Our recent study focused on characterizing observed variations in microbially induced gingival inflammation—resulting in three distinct clinical Inflammatory Responder Types (IRTs): High-IRT, Low-IRT, and Slow-IRT. Here, we applied a high-resolution temporal multiomic analysis during microbially induced inflammation in order to characterize the effects of localized oral inflammation on distant healthy tissues in young healthy adults. Our results highlight a nonlocalized subclinical effect with alterations in proinflammatory host mediators and an ecological shift toward dysbiosis within the subgingival microbiome in an IRT-dependent manner—despite maintained oral hygiene. Our results provide mechanistic insight into how healthy tissues within humans are influenced by distant localized inflammation and may ultimately become susceptible to disease. [ABSTRACT FROM AUTHOR]

To cause infection, pathogens must overcome bottlenecks imposed by the host immune system. These bottlenecks restrict the inoculum and largely determine whether pathogen exposure results in disease. Infection bottlenecks therefore quantify the effectiveness of immune barriers. Here, using a model of Escherichia coli systemic infection, we identify bottlenecks that tighten or widen with higher inoculum sizes, revealing that the efficacy of innate immune responses can increase or decrease with pathogen dose. We term this concept "dose scaling". During E. coli systemic infection, dose scaling is tissue specific, dependent on the lipopolysaccharide (LPS) receptor TLR4, and can be recapitulated by mimicking high doses with killed bacteria. Scaling therefore depends on sensing of pathogen molecules rather than interactions between the host and live bacteria. We propose that dose scaling quantitatively links innate immunity with infection bottlenecks and is a valuable framework for understanding how the inoculum size governs the outcome of pathogen exposure. [ABSTRACT FROM AUTHOR]

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are major regulators of innate and adaptive immune responses. DCs can be classified into plasmacytoid DCs and conventional DCs (cDCs) type 1 and 2. Murine and human cDC1 share the mRNA expression of XCR1. Murine studies indicated a specific role of the XCR1--XCL1 axis in the induction of immune responses. Here, we describe that human cDC1 can be distinguished into XCR1- and XCR1+ cDC1 in lymphoid as well as nonlymphoid tissues. Steady-state XCR1+ cDC1 display a preactivated phenotype compared to XCR1- cDC1. Upon stimulation, XCR1+ cDC1, but not XCR1- cDC1, secreted high levels of inflammatory cytokines as well as chemokines. This was associated with enhanced activation of NK cells mediated by XCR1+ cDC1. Moreover, XCR1+ cDC1 excelled in inhibiting replication of Influenza A virus. Further, under DC differentiation conditions, XCR1- cDC1 developed into XCR1+ cDC1. After acquisition of XCR1 expression, XCR1- cDC1 secreted comparable level of inflammatory cytokines. Thus, XCR1 is a marker of terminally differentiated cDC1 that licenses the antiviral effector functions of human cDC1, while XCR1- cDC1 seem to represent a late immediate precursor of cDC1. [ABSTRACT FROM AUTHOR]

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant--microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule- containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9- targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild- type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)- induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)- triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment. [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells which recognize a limited repertoire of ligands presented by the MHC class-I like molecule MR1. In addition to their key role in host protection against bacterial and viral pathogens, MAIT cells are emerging as potent anti-cancer effectors. With their abundance in human, unrestricted properties, and rapid effector functions MAIT cells are emerging as attractive candidates for immunotherapy. In the current study, we demonstrate that MAIT cells are potent cytotoxic cells, rapidly degranulating and inducing target cell death. Previous work from our group and others has highlighted glucose metabolism as a critical process for MAIT cell cytokine responses at 18 h. However, the metabolic processes supporting rapid MAIT cell cytotoxic responses are currently unknown. Here, we show that glucose metabolism is dispensable for both MAIT cell cytotoxicity and early (<3 h) cytokine production, as is oxidative phosphorylation. We show that MAIT cells have the machinery required to make (GYS-1) and metabolize (PYGB) glycogen and further demonstrate that that MAIT cell cytotoxicity and rapid cytokine responses are dependent on glycogen metabolism. In summary, we show that glycogen-fueled metabolism supports rapid MAIT cell effector functions (cytotoxicity and cytokine production) which may have implications for their use as an immunotherapeutic agent. [ABSTRACT FROM AUTHOR]

A hepatitis C virus (HCV) vaccine is urgently needed. Vaccine development has been hindered by HCV's genetic diversity, particularly within the immunodominant hypervariable region 1 (HVR1). Here, we developed a strategy to elicit broadly neutralizing antibodies to HVR1, which had previously been considered infeasible. We first applied a unique information theory-based measure of genetic distance to evaluate phenotypic relatedness between HVR1 variants. These distances were used to model the structure of HVR1's sequence space, which was found to have five major clusters. Variants from each cluster were used to immunize mice individually, and as a pentavalent mixture. Sera obtained following immunization neutralized every variant in a diverse HCVpp panel (n = 10), including those resistant to monovalent immunization, and at higher mean titers (1/ID50 = 435) than a glycoprotein E2 (1/ID50 = 205) vaccine. This synergistic immune response offers a unique approach to overcoming antigenic variability and may be applicable to other highly mutable viruses. [ABSTRACT FROM AUTHOR]

The first clinical efficacy trials of a broadly neutralizing antibody (bNAb) resulted in less benefit than expected and suggested that improvements are needed to prevent HIV infection. While considerable effort has focused on optimizing neutralization breadth and potency, it remains unclear whether augmenting the effector functions elicited by broadly neutralizing antibodies (bNAbs) may also improve their clinical potential. Among these effector functions, complement-mediated activities, which can culminate in the lysis of virions or infected cells, have been the least well studied. Here, functionally modified variants of the second-generation bNAb 10-1074 with ablated and enhanced complement activation profiles were used to examine the role of complement-associated effector functions. When administered prophylactically against simian-HIV challenge in rhesus macaques, more bNAb was required to prevent plasma viremia when complement activity was eliminated. Conversely, less bNAb was required to protect animals from plasma viremia when complement activity was enhanced. These results suggest that complement-mediated effector functions contribute to in vivo antiviral activity, and that their engineering may contribute to the further improvements in the efficacy of antibody-mediated prevention strategies. [ABSTRACT FROM AUTHOR]

Antibodies play a central role in the immune defense against SARS-CoV-2. Emerging evidence has shown that nonneutralizing antibodies are important for immune defense through Fc-mediated effector functions. Antibody subclass is known to affect downstream Fc function. However, whether the antibody subclass plays a role in anti-SARS-CoV-2 immunity remains unclear. Here, we subclass-switched eight human IgG1 anti-spike monoclonal antibodies (mAbs) to the IgG3 subclass by exchanging their constant domains. The IgG3 mAbs exhibited altered avidities to the spike protein and more potent Fc-mediated phagocytosis and complement activation than their IgG1 counterparts. Moreover, combining mAbs into oligoclonal cocktails led to enhanced Fc- and complement receptor-mediated phagocytosis, superior to even the most potent single IgG3 mAb when compared at equivalent concentrations. Finally, in an in vivo model, we show that opsonic mAbs of both subclasses can be protective against a SARS-CoV-2 infection, despite the antibodies being nonneutralizing. Our results suggest that opsonic IgG3 oligoclonal cocktails are a promising idea to explore for therapy against SARS-CoV-2, its emerging variants, and potentially other viruses. [ABSTRACT FROM AUTHOR]

Males of many species, ranging from humans to insects, are more susceptible than females to parasitic, fungal, bacterial, and viral infections. One mechanism that has been proposed to account for this difference is the immunocompetence handicap model, which posits that the greater infectious disease burden in males is due to testosterone, which drives the development of secondary male sex characteristics at the expense of suppressing immunity. However, emerging data suggest that cell-intrinsic (chromosome X and Y) sex-specific factors also may contribute to the sex differences in infectious disease burden. Using a murine model of influenza A virus (IAV) infection and a panel of chromosome Y (ChrY) consomic strains on the C57BL/6J background, we present data showing that genetic variation in ChrY influences IAV pathogenesis in males. Specific ChrY variants increase susceptibility to IAV in males and augment pathogenic immune responses in the lung, including activation of proinflammatory IL-17–producing γδ T cells, without affecting viral replication. In addition, susceptibility to IAV segregates independent of copy number variation in multicopy ChrY gene families that influence susceptibility to other immunopathological phenotypes, including survival after infection with coxsackievirus B3. These results demonstrate a critical role for genetic variation in ChrY in regulating susceptibility to infectious disease. [ABSTRACT FROM AUTHOR]

The article offers inoramtion related to the development and commercialization of conjugate vaccines to eliminate bacterial meningitis disease. It is noted that after immunization, conjugate vaccines are taken up by dendritic cells and transported to lymph nodes to induce immune response to engage both B and T cells. It also discusses way to improve immunogenicity of vaccine by increasing the number of covalent junctions between protein and polysaccharide.

The current view of nucleic acid-mediated innate immunity is that binding of intracellular sensors to nucleic acids is sufficient for their activation. Here, we report that endocytosis of virus or foreign DNA initiates a priming signal for the DNA sensor cyclic GMP-AMP synthase (cGAS)-mediated innate immune response. Mechanistically, viral infection or foreign DNA transfection triggers recruitment of the spleen tyrosine kinase (SYK) and cGAS to the endosomal vacuolar H+ pump (V-ATPase), where SYK is activated and then phosphorylates human cGASY214/215 (mouse cGasY200/201) to prime its activation. Upon binding to DNA, the primed cGAS initiates robust cGAMP production and mediator of IRF3 activation/stimulator of interferon genes-dependent innate immune response. Consistently, blocking the V-ATPase-SYK axis impairs DNA virusand transfected DNA-induced cGAMP production and expression of antiviral genes. Our findings reveal that V-ATPase-SYK-mediated tyrosine phosphorylation of cGAS following endocytosis of virus or other cargos serves as a priming signal for cGAS activation and innate immune response. [ABSTRACT FROM AUTHOR]

The pregnant uterus is an immunologically rich organ, with dynamic changes in the inflammatory milieu and immune cell function underlying key stages of pregnancy. Recent studies have implicated dysregulated expression of the interleukin-1 (IL-1) family cytokine, IL-33, and its receptor, ST2, in poor pregnancy outcomes in women, including recurrent pregnancy loss, preeclampsia, and preterm labor. How IL-33 supports pregnancy progression in vivo is not well understood. Here, we demonstrate that maternal IL-33 signaling critically regulates uterine tissue remodeling and immune cell function during early pregnancy in mice. IL-33-deficient dams exhibit defects in implantation chamber formation and decidualization, and abnormal vascular remodeling during early pregnancy. These defects coincide with delays in early embryogenesis, increased resorptions, and impaired fetal and placental growth by late pregnancy. At a cellular level, myometrial fibroblasts, and decidual endothelial and stromal cells, are the main IL-33+ cell types in the uterus during decidualization and early placentation, whereas ST2 is expressed by uterine immune populations associated with type 2 immune responses, including ILC2s, Tregs, CD4+ T cells, M2- and cDC2-like myeloid cells, and mast cells. Early pregnancy defects in IL-33-deficient dams are associated with impaired type 2 cytokine responses by uterine lymphocytes and fewer Arginase-1+ macrophages in the uterine microenvironment. Collectively, our data highlight a regulatory network, involving crosstalk between IL-33-producing nonimmune cells and ST2+ immune cells at the maternal-fetal interface, that critically supports pregnancy progression in mice. This work has the potential to advance our understanding of how IL-33 signaling may support optimal pregnancy outcomes in women. [ABSTRACT FROM AUTHOR]

Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency in vivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both in vitro and in vivo. However, T cell priming in vivo required Batf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency. [ABSTRACT FROM AUTHOR]

Immunotherapy has had a tremendous impact on cancer treatment in the past decade, with hitherto unseen responses at advanced and metastatic stages of the disease. However, the aggressive brain tumor glioblastoma (GBM) is highly immunosuppressive and remains largely refractory to current immunotherapeutic approaches. The stimulator of interferon genes (STING) DNA sensing pathway has emerged as a next-generation immunotherapy target with potent local immune stimulatory properties. Here, we investigated the status of the STING pathway in GBM and the modulation of the brain tumor microenvironment (TME) with the STING agonist ADU-S100. Our data reveal the presence of STING in human GBM specimens, where it stains strongly in the tumor vasculature.We show that human GBM explants can respond to STING agonist treatment by secretion of inflammatory cytokines. In murine GBM models, we show a profound shift in the tumor immune landscape after STING agonist treatment, with massive infiltration of the tumor-bearing hemisphere with innate immune cells including inflammatory macrophages, neutrophils, and natural killer (NK) populations. Treatment of established murine intracranial GL261 and CT-2A tumors by biodegradable ADU-S100–loaded intracranial implants demonstrated a significant increase in survival in both models and long-term survival with immune memory in GL261. Responses to treatment were abolished by NK cell depletion. This study reveals therapeutic potential and deep remodeling of the TME by STING activation in GBM and warrants further examination of STING agonists alone or in combination with other immunotherapies such as cancer vaccines, chimeric antigen receptor T cells, NK therapies, and immune checkpoint blockade. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICIs) are essential components of the cancer therapeutic armamentarium. While ICIs have demonstrated remarkable clinical responses, they can be accompanied by immune-related adverse events (irAEs). These inflammatory side effects are of unclear etiology and impact virtually all organ systems, with the most common being sites colonized by the microbiota such as the skin and gastrointestinal tract. Here, we establish a mouse model of commensal bacteria-driven skin irAEs and demonstrate that immune checkpoint inhibition unleashes commensal-specific inflammatory T cell responses. These aberrant responses were dependent on production of IL-17 by commensal-specific T cells and induced pathology that recapitulated the cutaneous inflammation seen in patients treated with ICIs. Importantly, aberrant T cell responses unleashed by ICIs were sufficient to perpetuate inflammatory memory responses to the microbiota months following the cessation of treatment. Altogether, we have established a mouse model of skin irAEs and reveal that ICIs unleash aberrant immune responses against skin commensals, with long-lasting inflammatory consequences. [ABSTRACT FROM AUTHOR]

The epidermis is the outermost layer of the skin and the body's primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen Staphylococcus aureus in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to S. aureus DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system. [ABSTRACT FROM AUTHOR]

Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC- 101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Treg) can impede antitumor immunity and currently represent a major obstacle to effective cancer immunotherapy. Targeting tumor-infiltrating regulatory Treg while sparing systemic Treg represents an optimal approach to this problem. Here, we provide evidence that the interleukin 23 receptor (IL23R) expressed by tumor-infiltrating Treg promotes suppressive activity. Disruption of the IL23R results in increased responsiveness of destabilized Treg to the IL12 cytokine, the production of γ-interferon, and the recruitment of CD8 T cells that inhibit tumor growth. Since the Treg destabilization pathway that is initiated by IL23R blockade is distinct and independent from the destabilization pathway coupled to glucocorticoid-induced TNFRrelated protein (GITR) activation, we examined the impact of the coordinate induction of the two destabilization pathways on antitumor immune responses. Combined GITR and IL23R antibody treatment of mice inoculated with MC38 tumors resulted in robust and synergistic antitumor responses. These findings indicate that the delineation of independent Treg destabilization pathways may allow improved approaches to the development of combination immunotherapy for cancers. [ABSTRACT FROM AUTHOR]

Despite more than 300,000 rVSVΔG-ZEBOV-glycoprotein (GP) vaccine doses having been administered during Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC) between 2018 and 2020, seroepidemiologic studies of vaccinated Congolese populations are lacking. This study examines the anti-body response at 21 d and 6 mo postvaccination after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC. We conducted a longitudinal cohort study of 608 rVSVΔG-ZEBOV-GP-vaccinated individuals during an EVD outbreak in North Kivu Province, DRC. Participants provided questionnaires and blood samples at three study visits (day 0, visit 1; day 21, visit 2; and month 6, visit 3). Anti-GP immunoglobulin G (IgG) antibody titers were measured in serum by the Filovirus Animal Nonclinical Group anti-Ebola virus GP IgG enzyme-linked immunosorbent assay. Antibody response was defined as an antibody titer that had increased fourfold from visit 1 to visit 2 and was above four times the lower limit of quantification at visit 2; antibody persistence was defined as a similar increase from visit 1 to visit 3. We then examined demographics for associations with follow-up antibody titers using generalized linear mixed models. A majority of the sample, 87.2%, had an anti-body response at visit 2, and 95.6% demonstrated antibody persistence at visit 3. Being female and of young age was predictive of a higher antibody titer postvaccination. Antibody response and persistence after Ebola vaccination was robust in this cohort, confirming findings from outside of the DRC. [ABSTRACT FROM AUTHOR]

SAMD9 and SAMD9L (SAMD9/9L) are antiviral factors and tumor suppressors, playing a critical role in innate immune defense against poxviruses and the development of myeloid tumors. SAMD9/9L mutations with a gain-of-function (GoF) in inhibiting cell growth cause multisystem developmental disorders including many pediatric myelodysplastic syndromes. Predicted to be multidomain proteins with an architecture like that of the NOD-like receptors, SAMD9/9L molecular functions and domain structures are largely unknown. Here, we identified a SAMD9/9L effector domain that functions by binding to double-stranded nucleic acids (dsNA) and determined the crystal structure of the domain in complex with DNA. Aided with precise mutations that differentially perturb dsNA binding, we demonstrated that the antiviral and antiproliferative functions of the wild-type and GoF SAMD9/9L variants rely on dsNA binding by the effector domain. Furthermore, we showed that GoF variants inhibit global protein synthesis, reduce translation elongation, and induce proteotoxic stress response, which all require dsNA binding by the effector domain. The identification of the structure and function of a SAMD9/9L effector domain provides a therapeutic target for SAMD9/9Lassociated human diseases. [ABSTRACT FROM AUTHOR]

The axis of platelet-derived growth factor (PDGF) and PDGF receptor-beta (PDGFRß) plays prominent roles in cell growth and motility. In addition, PDGF-D enhances human natural killer (NK) cell effector functions when binding to the NKp44 receptor. Here, we report an additional but previously unknown role of PDGF-D, whereby it mediates interleukin-15 (IL-15)-induced human NK cell survival but not effector functions via its binding to PDGFRß but independent of its binding to NKp44. Resting NK cells express no PDGFRß and only a low level of PDGF-D, but both are significantly up-regulated by IL-15, via the nuclear factor B signaling pathway, to promote cell survival in an autocrine manner. Both ectopic and IL-15-induced expression of PDGFRß improves NK cell survival in response to treatment with PDGF-D. Our results suggest that the PDGF-D2PDGFRß signaling pathway is a mechanism by which IL-15 selectively regulates the survival of human NK cells without modulating their effector functions. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease. [ABSTRACT FROM AUTHOR]

Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)–containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C ) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the β2-β3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C . Structure-based mutations that disrupt AvrRps4C –RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY–Wbox DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity. [ABSTRACT FROM AUTHOR]

Insects respond to microbial infection by the rapid and transient expression of several genes encoding antibacterial peptides. In this paper we describe a powerful technique, two-dimensional difference gel electrophoresis, that, when combined with mass spectrometry. can be used to study the immune response of Drosophila melano-gaster at the protein level. By comparatively analyzing the hemo-lymph proteome of 2,000 third-instar Drosophila larvae, we identified 10 differential proteins that appear in the fruit fly hemolymph very early after an immune-challenge with lipopolysaccharides. These proteins can be assigned to the immune response, because they are not induced after sterile injury. Reduction of intergel variability or quantification problems related to conventional two-dimensional electrophoresis and improvement of image analysis were achieved by the use of two fluorescent dyes to label the two different protein samples. Some of the immune-induced proteins, such as thioester- containing protein 2, can be assigned to specific aspects of the immune response; others were already reported as being involved in stress response. An immune-induced protein (CG18594) is homologous to a mammalian serine protease inhibitor that mediates the mitogen-activated protein kinase and the NF-κB signaling pathways. In addition, a number of proteins that had not been associated with the immune response before were isolated and identified, and some of these were still present in the hemolymph 4 h after injury. Determining the function of all of these immune-induced proteins represents an exciting challenge for increasing our knowledge of insect immunity. [ABSTRACT FROM AUTHOR]

Invariant natural killer T (iNKT) cells play important roles in regulating immune responses. Based on cytokine profiling and key transcriptional factors, iNKT cells are classified into iNKT1, iNKT2, and iNKT17 subsets. However, whether the development and functions of these subsets are controlled by distinct mechanisms remains unclear. Here, we show that forkhead box protein O1 (Foxo1) promotes differentiation of iNKT1 and iNKT2 cells but not iNKT17 cells because of its distinct contributions to IL7R expression in these subsets. Nuclear Foxo1 is essential for Il7r expression in iNKT1 and iNKT2 cells at early stages of differentiation but is dispensable in iNKT17 cells. ROR?t, instead of Foxo1, promotes IL7R expression in iNKT17 cells. Additionally, Foxo1 is required for the effector function of iNKT1 and iNKT2 cells but not iNKT17 cells. Cytoplasmic Foxo1 promotes activation of mTORC1 in iNKT1 and iNKT2 cells through inhibiting TSC1-TSC2 interaction, whereas it is dispensable for mTORC1 activation in iNKT17 cells. iNKT17 cells display distinct metabolic gene expression patterns from iNKT1 and iNKT2 cells that match their different functional requirements on Foxo1. Together, our results demonstrate that iNKT cell subsets differ in their developmental and functional requirements on Foxo1. [ABSTRACT FROM AUTHOR]

Cellular ionic concentrations are a central factor orchestrating host innate immunity, but no pathogenic mechanism that perturbs host innate immunity by directly targeting metal ions has yet been described. Here, we report a unique virulence strategy of Yersinia pseudotuberculosis (Yptb) involving modulation of the availability of Mn2+, an immunostimulatory metal ion in host cells. We showed that the Yptb type VI secretion system (T6SS) delivered a micropeptide, TssS, into host cells to enhance its virulence. The mutant strain lacking TssS (ΔtssS) showed substantially reduced virulence but induced a significantly stronger host innate immune response, indicating an antagonistic role of this effector in host antimicrobial immunity. Subsequent studies revealed that TssS is a Mn2+-chelating protein and that its Mn2+-chelating ability is essential for the disruption of host innate immunity. Moreover, we showed that Mn2+ enhances the host innate immune response to Yptb infection by activating the stimulator of interferon genes (STING)-mediated immune response. Furthermore, we demonstrated that TssS counteracted the cytoplasmic Mn2+ increase to inhibit the STING-mediated innate immune response by sequestering Mn2+. Finally, TssS-mediated STING inhibition sabotaged bacterial clearance in vivo. These results reveal a previously unrecognized bacterial immune evasion strategy involving modulation of the bioavailability of intracellular metal ions and provide a perspective on the role of the T6SS in pathogenesis. [ABSTRACT FROM AUTHOR]

Morbidity and mortality rates from seasonal and pandemic influenza occur disproportionately in high-risk groups, including Indigenous people globally. Although vaccination against influenza is recommended for those most at risk, studies on immune responses elicited by seasonal vaccines in Indigenous populations are largely missing, with no data available for Indigenous Australians and only one report published on antibody responses in Indigenous Canadians. We recruited 78 Indigenous and 84 non-Indigenous Australians vaccinated with the quadrivalent influenza vaccine into the Looking into InFluenza T cell immunity - Vaccination cohort study and collected blood to define baseline, early (day 7), and memory (day 28) immune responses. We performed in-depth analyses of T and B cell activation, formation of memory B cells, and antibody profiles and investigated host factors that could contribute to vaccine responses. We found activation profiles of circulating T follicular helper type-1 cells at the early stage correlated strongly with the total change in antibody titers induced by vaccination. Formation of influenza-specific hemagglutinin-binding memory B cells was significantly higher in seroconverters compared with nonseroconverters. In-depth antibody characterization revealed a reduction in immunoglobulin G3 before and after vaccination in the Indigenous Australian population, potentially linked to the increased frequency of the G3m21* allotype. Overall, our data provide evidence that Indigenous populations elicit robust, broad, and prototypical immune responses following immunization with seasonal inactivated influenza vaccines. Our work strongly supports the recommendation of influenza vaccination to protect Indigenous populations from severe seasonal influenza virus infections and their subsequent complications. [ABSTRACT FROM AUTHOR]

In multicellular organisms, antiviral defense mechanisms evoke a reliable collective immune response despite the noisy nature of biochemical communication between tissue cells. A molecular hub of this response, the interferon I receptor (IFNAR), discriminates between ligand types by their affinity regardless of concentration. To understand how ligand type can be decoded robustly by a single receptor, we frame ligand discrimination as an informationtheoretic problem and systematically compare the major classes of receptor architectures: allosteric, homodimerizing, and heterodimerizing. We demonstrate that asymmetric heterodimers achieve the best discrimination power over the entire physiological range of local ligand concentrations. This design enables sensing of ligand presence and type, and it buffers against moderate concentration fluctuations. In addition, receptor turnover, which drives the receptor system out of thermodynamic equilibrium, allows alignment of activation points for ligands of different affinities and thereby makes ligand discrimination practically independent of concentration. IFNAR exhibits this optimal architecture, and our findings thus suggest that this specialized receptor can robustly decode digital messages carried by its different ligands. [ABSTRACT FROM AUTHOR]

Perturbation of lung homeostasis is frequently associated with progressive and fatal respiratory diseases, such as pulmonary fibrosis. Leucine-rich repeat kinase 2 (LRRK2) is highly expressed in healthy lungs, but its functions in lung homeostasis and diseases remain elusive. Herein,we showed that LRRK2 expression was clearly reduced in mammalian fibrotic lungs, and LRRK2-deficient mice exhibited aggravated bleomycin-induced pulmonary fibrosis. Furthermore, we demonstrated that in bleomycin-treated mice, LRRK2 expression was dramatically decreased in alveolar type II epithelial (AT2) cells, and its deficiency resulted in profound dysfunction of AT2 cells, characterized by impaired autophagy and accelerated cellular senescence. Additionally, LRRK2-deficient AT2 cells showed a higher capacity of recruiting profibrotic macrophages via the CCL2/CCR2 signaling, leading to extensive macrophage-associated profibrotic responses and progressive pulmonary fibrosis. Taken together, our study demonstrates that LRRK2 plays a crucial role in preventing AT2 cell dysfunction and orchestrating the innate immune responses to protect against pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

Male and female animals exhibit differences in infection outcomes. One possible source of sexually dimorphic immunity is the sexspecific costs of immune activity or pathology, but little is known about the independent effects of immune-versus microbe-induced pathology and whether these may differ for the sexes. Here, by measuring metabolic and physiological outputs in Drosophila melanogaster with wild-type and mutant immune responses, we test whether the sexes are differentially impacted by these various sources of pathology and identify a critical regulator of this difference. We find that the sexes exhibit differential immune activity but similar bacteria-derived metabolic pathology. We show that femalespecific immune-inducible expression of PGRP-LB, a negative regulator of the immune deficiency (IMD) pathway, enables females to reduce immune activity in response to reductions in bacterial numbers. In the absence of PGRP-LB, females are more resistant to infection, confirming the functional importance of this regulation and suggesting that female-biased immune restriction comes at a cost. [ABSTRACT FROM AUTHOR]

Given the role of myeloid cells in T cell activation and in the antitumor response, targeting checkpoint molecules expressed on this population represents a promising strategy to augment antitumor immunity. However, myeloid checkpoints that can be effectively used as immunotherapy targets are still lacking. Here, we demonstrate the therapeutic potential of targeting the myeloid receptors Siglec-7 and Siglec-9 in vivo. By using a humanized immunocompetent murine model, we demonstrate that human Siglec-7 and Siglec-9, in addition to the murine homolog Siglec-E, inhibit the endogenous antitumor immune response, as well as the response to tumor-targeting and immune checkpoint inhibiting antibodies in vivo. The impact of these Siglecs on tumor progression is highly dependent on the anatomical distribution of the tumor and, as a consequence, the local tumor microenvironment, as tumors with a more immune-suppressive tumor microenvironment are less sensitive to Siglec perturbation. Finally, to assess the potential of these two receptors as targets for immunotherapy, we developed Fc engineered blocking antibodies to Siglec-7 and Siglec-9 and demonstrate that Siglec-7 and Siglec-9 blockade can significantly reduce tumor burden in vivo, demonstrating the therapeutic potential of targeting these two receptors. [ABSTRACT FROM AUTHOR]

The ongoing COVID-19 pandemic has caused an unprecedented global health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. Subversion of host protein synthesis is a common strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to shut down host protein synthesis and that SARS-CoV-2 nonstructural protein NSP14 exerts this activity. We show that the translation inhibition activity of NSP14 is conserved in human coronaviruses. NSP14 is required for virus replication through contribution of its exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. Mutations in the ExoN or N7-MTase active sites of SARS-CoV-2 NSP14 abolish its translation inhibition activity. In addition, we show that the formation of NSP14−NSP10 complex enhances translation inhibition executed by NSP14. Consequently, the translational shutdown by NSP14 abolishes the type I interferon (IFN-I)-dependent induction of interferon-stimulated genes (ISGs). Together, we find that SARS-CoV-2 shuts down host innate immune responses via a translation inhibitor, providing insights into the pathogenesis of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Loss of the retinal pigment epithelium (RPE) because of dysfunction or disease can lead to blindness in humans. Harnessing the intrinsic ability of the RPE to self-repair is an attractive therapeutic strategy; however, mammalian RPE is limited in its regenerative capacity. Zebrafish possess tremendous intrinsic regenerative potential in ocular tissues, including the RPE, but little is known about the mechanisms driving RPE regeneration. Here, utilizing transgenic and mutant zebrafish lines, pharmacological manipulations, transcriptomics, and imaging analyses, we identified elements of the immune response as critical mediators of intrinsic RPE regeneration. After genetic ablation, the RPE express immune-related genes, including leukocyte recruitment factors such as interleukin 34. We demonstrate that macrophage/microglia cells are responsive to RPE damage and that their function is required for the timely progression of the regenerative response. These data identify the molecular and cellular underpinnings of RPE regeneration and hold significant potential for translational approaches aimed toward promoting a pro-regenerative environment in mammalian RPE. [ABSTRACT FROM AUTHOR]

Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD. [ABSTRACT FROM AUTHOR]

Intracarotid arterial hyperosmolar mannitol (ICAHM) blood-brain barrier disruption (BBBD) is effective and safe for delivery of therapeutics for central nervous system malignancies. ICAHM osmotically alters endothelial cells and tight junction integrity to achieve BBBD. However, occurrence of neuroinflammation following hemispheric BBBD by ICAHM remains unknown. Temporal proteomic changes in rat brains following ICAHM included increased damage-associated molecular patterns, cytokines, chemokines, trophic factors, and cell adhesion molecules, indicative of a sterile inflammatory response (SIR). Proteomic changes occurred within 5 min of ICAHM infusion and returned to baseline by 96 h. Transcriptomic analyses following ICAHM BBBD further supported an SIR. Immunohistochemistry revealed activated astrocytes, microglia, and macrophages. Moreover, proinflammatory proteins were elevated in serum, and proteomic and histological findings from the contralateral hemisphere demonstrated a less pronounced SIR, suggesting neuroinflammation beyond regions of ICAHM infusion. Collectively, these results demonstrate ICAHM induces a transient SIR that could potentially be harnessed for neuroimmunomodulation. [ABSTRACT FROM AUTHOR]

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130-4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623-630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341-3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steadystate barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation. [ABSTRACT FROM AUTHOR]

Macrophages are the key regulator of T-cell responses depending on their activation state. C-C motif chemokine receptor-like 2 (CCRL2), a nonsignaling atypical receptor originally cloned from LPS-activated macrophages, has recently been shown to regulate immune responses under several inflammatory conditions. However, whether CCRL2 influences macrophage function and regulates tumor immunity remains unknown. Here, we found that tumoral CCRL2 expression is a predictive indicator of robust antitumor T-cell responses in human cancers. CCRL2 is selectively expressed in tumor-associated macrophages (TAM) with immunostimulatory phenotype in humans and mice. Conditioned media from tumor cells could induce CCRL2 expression in macrophages primarily via TLR4, which is negated by immunosuppressive factors. Ccrl2-/- mice exhibit accelerated melanoma growth and impaired antitumor immunity characterized by significant reductions in immunostimulatory macrophages and T-cell responses in tumor. Depletion of CD8+ T cells or macrophages eliminates the difference in tumor growth between WT and Ccrl2-/- mice. Moreover, CCRL2 deficiency impairs immunogenic activation of macrophages, resulting in attenuated antitumor T-cell responses and aggravated tumor growth in a coinjection tumor model. Mechanically, CCRL2 interacts with TLR4 on the cell surface to retain membrane TLR4 expression and further enhance its downstream Myd88-NF-κB inflammatory signaling in macrophages. Similarly, Tlr4-/- mice exhibit reduced CCRL2 expression in TAM and acceleratedmelanoma growth. Collectively, our study reveals a functional role of CCRL2 in activating immunostimulatory macrophages, thereby potentiating antitumor T-cell response and tumor rejection, and suggests CCLR2 as a potential biomarker candidate and therapeutic target for cancer immunotherapy. [ABSTRACT FROM AUTHOR]