TLR-dependent phagosome tubulation in dendritic cells promotes phagosome cross-talk to optimize MHC-II antigen presentation

Dendritic cells (DCs) phagocytose large particles like bacteria at sites of infection and progressively degrade them within maturing phagosomes. Phagosomes in DCs are also signaling platforms for pattern recognition receptors, such as Toll-like receptors (TLRs), and sites for assembly of cargo-derived peptides with major histocompatibility complex class II (MHC-II) molecules. Although TLR signaling from phagosomes stimulates presentation of phagocytosed antigens, the mechanisms underlying this enhancement and the cell surface delivery of MHC-II–peptide complexes from phagosomes are not known. We show that in DCs, maturing phagosomes extend numerous long tubules several hours after phagocytosis. Tubule formation requires an intact microtubule and actin cytoskeleton and MyD88-dependent phagosomal TLR signaling, but not phagolysosome formation or extensive proteolysis. In contrast to the tubules that emerge from endolysosomes after uptake of soluble ligands and TLR stimulation, the late-onset phagosomal tubules are not essential for delivery of phagosome-derived MHC-II–peptide complexes to the plasma membrane. Rather, tubulation promotes MHC-II presentation by enabling maximal cargo transfer among phagosomes that bear a TLR signature. Our data show that phagosomal tubules in DCs are functionally distinct from those that emerge from lysosomes and are unique adaptations of the phagocytic machinery that facilitate cargo exchange and antigen presentation among TLR-signaling phagosomes.Professional phagocytes take up large particles, such as bacteria, by phagocytosis and submit them to an increasingly harsh environment during phagosome maturation (1). Phagocytes concomitantly alert the immune system that an invader is present via signaling programs initiated by pattern recognition receptors, such as Toll-like receptors (TLRs) (2). Conventional dendritic cells (DCs) also alter and optimize phagosome maturation and TLR-signaling programs to preserve bacterial antigens for loading onto MHC class I and class II (MHC-II) molecules and optimize cytokine secretion to stimulate and direct T-cell responses to the invading agent (3, 4). DC presentation of soluble antigen is facilitated by TLR-driven tubulation of lysosomes that harbor MHC-II–peptide complexes and by consequent fusion of tubulovesicular structures with the plasma membrane (5–7); however, little is known about the mechanism by which signaling pathways influence the formation or presentation of phagosome-derived MHC-II–peptide complexes, key processes in the adaptive immunity to bacterial pathogens.TLRs respond to microbial ligands at the plasma membrane and in intracellular stores (8). TLR stimulation at the plasma membrane, endosomes, or phagosomes elicits distinct signaling pathways via two sets of adaptors, TIRAP (or MAL)-MyD88 and TRAM-TRIF (8, 9), which induce proinflammatory cytokine secretion and other downstream responses. TLRs such as TLR2 and TLR4 are recruited to macrophage and DC phagosomes at least partly from an intracellular pool (10–13), and signal autonomously from phagosomes independent of plasma membrane TLRs (11, 14, 15). Autonomous phagosomal signaling from TLRs or Fcγ receptors enhances the degradation of phagocytosed proteins and assembly of MHC-II with their derived peptides (14–16). Phagosomal TLR signaling has been proposed to also promote the reorganization of phagosome-derived MHC-II-enriched compartments (MIICs) to favor the delivery of MHC-II–peptide complexes to the plasma membrane (17), analogous to TLR-stimulated formation of tubules from MIICs/lysosomes (18–20) that fuse with the plasma membrane (7) and extend toward the immunologic synapse with T cells (5). Tubules emerge from phagosomes in macrophages shortly after phagocytosis and likely function in membrane recycling during early phagosome maturation stages (21–23), but tubules at later stages that might facilitate the presentation of phagosome-derived MHC-II–peptide complexes have not been reported previously. Moreover, a role for TLR signaling in formation of phagosome-derived tubules has not been established.Herein we show that in DCs, maturing phagosomes undergo extensive tubulation up to several hours after phagocytosis, and that tubulation requires TLR and MyD88 signaling and an intact actin and microtubule cytoskeleton. Unlike lysosome tubulation, phagosome tubulation is not essential for MHC-II–peptide transport to the cell surface. Rather, it contributes to content exchange among phagosomes that carry a TLR signature, and thereby enhances presentation of phagocytosed antigens from potential pathogens.     

Lytic immune synapse function requires filamentous actin deconstruction by Coronin 1A

Lytic immune effector function depends upon directed secretion of cytolytic granules at the immunological synapse (IS) and requires dynamic rearrangement of filamentous (F)-actin. Coronin 1A (Coro1A) is the hematopoietic-specific member of the Coronin family of actin regulators that promote F-actin disassembly. Here, we show that Coro1A is required for natural killer (NK) cell cytotoxic function in two human NK cell lines and ex vivo cells from a Coro1A-deficient patient. Using superresolution nanoscopy to probe the IS, we demonstrate that Coro1A promotes the deconstruction of F-actin density that facilitates effective delivery of lytic granules to the IS. Thus, we show, for the first time to our knowledge, a critical role for F-actin deconstruction in cytotoxic function and immunological secretion and identify Coro1A as its mediator.Natural killer (NK) cell cytotoxicity is a finely controlled process that integrates signals from activating and inhibitory receptors to eliminate virally infected and tumorigenic cells sensitively and specifically. The importance for NK cells in immune function is underscored by the severe virus infections and malignancies suffered by patients with NK cell deficiency (1). A dynamic filamentous (F)-actin cytoskeleton is required for NK cell cytotoxicity because disruption of F-actin polymerization by pharmacological inhibitors or mutation of actin-nucleating factors results in impaired NK cell function (2–5). Actin nucleators, such as actin-related proteins 2 and 3 complex (Arp2/3), Wiskott–Aldrich syndrome protein (WASp), WIP, DOCK8, and WAVE2, serve well-defined critical roles in the formation and function of the NK cell immunological synapse (IS) (4–10).Killing of a susceptible target follows tightly regulated steps of NK cell immune synapse formation and lytic granule exocytosis (3). Although cortical F-actin has long been considered a barrier to exocytosis of granule-like organelles (11) in some cell types, ligation of NK cell activating and adhesion receptors results in the formation of conduits in F-actin that permit and actually facilitate NK cell degranulation (12–14). This finding suggests that fine regulation and deconstruction of the synaptic F-actin meshwork is required for the formation of granule-permissive–sized clearances (12).Coronin 1A (Coro1A) is the hematopoietic cell-specific isoform of the highly conserved Coronin family of actin regulators. Coronins contain a series of WD-repeat domains that form an F-actin–binding β-propeller domain, and thus bind F-actin directly (15–17). In addition, Coro1A binds to and inhibits the Arp2/3 complex (17, 18) required for actin branching and can enhance the activity of cofilin to promote actin disassembly in in vitro reconstituted systems (19–21). Coro1A localizes with actin-rich structures in immune cells, including phagocytic cups in neutrophils and macrophages, and at the leading edge of T cells (22–25). T cells from Coronin 1^−/− mice have defects in migration and cell survival attributed to impaired T-cell receptor signaling, Ca²⁺ flux, Rac activation, and subcellular Arp2/3 localization (26–28). Mutations in Coro1A lead to T⁻B⁺NK⁺ combined immunodeficiency and susceptibility to severe viral infections, including life-threatening varicella infection and EBV-driven lymphoproliferation (26, 29, 30).By manipulating expression of Coro1A in human NK cells, we show that Coro1A is required for cytotoxic function. Using superresolution nanoscopy, we define a requirement for Coro1A in F-actin deconstruction and subsequent delivery of lytic granules to the synaptic membrane. In addition, we have specifically evaluated cytotoxic function in a Coro1A-deficient patient and find that NK cell function is severely impaired. Further, we demonstrate the same F-actin structural defect in patient cells as in two Coro1A-deficient cell lines. Thus, with superresolution imaging, we identify, for the first time to our knowledge, a critical role for actin deconstruction in immunity and human host defense.     

Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94

Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94–NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor–ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A⁺ NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR⁺ and NKG2A⁺ NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I–bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I.Natural killer (NK) cells play an important role in the immune response to viral infections and cancer. Their responses are determined by signals integrated from activating and inhibitory receptor–ligand interactions (1). In many situations inhibitory signals dominate activating signals. Therefore, releasing NK cells from inhibition is an important mechanism of enhancing their response to target cells. Inhibitory interactions are mediated by receptors for self-MHC class I. Most species have at least two discrete gene families of inhibitory receptors for MHC class I: the CD94–NKG2A C-type lectin-like receptor system and either the related Ly49 family of receptors or the unrelated killer cell Ig-like receptors (KIR) (2). The KIR family is important in humans and other primates, having undergone extensive diversification under positive selection. In contrast, the CD94–NKG2A system has remained relatively well conserved across the species with orthologous genes in primates and a closely related functional homolog in rodents (3, 4). Consistent with the coevolution of these families and their MHC class I ligands, KIR bind polymorphic MHC class I, HLA-A, -B, and -C molecules, whereas CD94–NKG2A binds the conserved oligomorphic HLA-E molecule or the rodent homolog Qa-1 (5, 6).Both receptor families are important in the immune response to viral infections. KIR are genetic determinants in the outcome of both HIV and hepatitis C virus (HCV) infection (7–10). Expression of CD94–NKG2A is up-regulated on NK cells in HIV and HCV infection and in the latter has been associated with a poor response to treatment (11, 12). Furthermore NKG2A⁺ NK cell clones lyse vaccinia-infected targets (13), and CD94 is important in clearing mouse pox infection (14). Both KIR and CD94–NKG2A respond to MHC class I down-regulation. One hypothesis is that the KIR have evolved to recognize MHC class I-specific down-regulation (15). However, because the majority of MHC class I leader peptides bind HLA-E and are inhibitory for NKG2A, the CD94–NKG2A system also is able to recognize down-regulation of most MHC class I alleles. It has been shown that KIR⁺ NK cells can be modulated by changes in the peptide bound by MHC class I, which confers additional functionality on the KIR system (16–18). In particular peptide antagonism is a potent mechanism for activating KIR⁺ NK cells (19, 20). The CD94–NKG2A receptor also is peptide selective, with receptor binding being particularly influenced by residues 5, 6, and 8 of the peptide bound by HLA-E (21–23). These residues interact primarily with the nonsignaling CD94 moiety, which occupies the majority of the HLA-E–binding interface. CD94–NKG2A seems to be a target for viral escape, with peptides derived from CMV, HCV, HIV, and EBV binding HLA-E and subsequently inhibiting NK cells (24–27). Viral peptides that inhibit at KIR also are identifiable (28), but their relevance likely is limited to the subset of individuals who have the relevant peptide-binding MHC class I allele. Understanding differences in how the KIR and NKG2 systems respond to peptide may be important for interpreting their roles in the immune response to viral infections and tumors. Therefore we explored how HLA-E–bound peptide can influence NK cell reactivity.     

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispair-containing DNA substrates. A mixture of MutS homolog 2 (Msh2)–MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5′ or 3′ strand interruption with different efficiencies. The Msh2–MutS homolog 3 mispair recognition protein could substitute for the Msh2–Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1–postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5′ or 3′ strand interruption occurred by mispair binding-dependent 5′ excision and subsequent resynthesis with excision tracts of up to ∼2.9 kb occurring during the repair of the substrate with a 3′ strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.DNA mismatch repair (MMR) is a critical DNA repair pathway that is coupled to DNA replication in eukaryotes where it corrects misincorporation errors made during DNA replication (1–9). This pathway prevents mutations and acts to prevent the development of cancer (10, 11). MMR also contributes to gene conversion by repairing mispaired bases that occur during the formation of recombination intermediates (3, 4, 12). Finally, MMR acts to suppress recombination between divergent but homologous DNA sequences, thereby preventing the formation of genome rearrangements that can result from nonallelic homologous recombination (4, 13–15).Our knowledge of the mechanism of eukaryotic MMR comes from several general lines of investigation (3–9). Studies of bacterial MMR have provided a basic mechanistic framework for comparative studies (5). Genetic and cell-biology studies, primarily in Saccharomyces cerevisiae, have identified eukaryotic MMR genes, provided models for how their gene products define MMR pathways, and elucidated some of the details of how MMR pathways interact with replication (1–4). Reconstitution studies, primarily in human systems, have identified some of the catalytic features of eukaryotic MMR (7–9, 16, 17). Biochemical and structural studies of S. cerevisiae and human MMR proteins have provided information about the function of individual MMR proteins (6–9).In eukaryotic MMR, mispairs are bound by MutS homolog 2 (Msh2)–MutS homolog 6 (Msh6) and Msh2–MutS homolog 3 (Msh3), two partially redundant complexes of MutS-related proteins (3, 4, 18, 19). These complexes recruit a MutL-related complex, called MutL homoloh 1 (Mlh1)–postmeiotic segregation 1 (Pms1) in S. cerevisiae and Mlh1–postmeiotic segregation 2 (Pms2) in human and mouse (3, 4, 20–23). The Mlh1–Pms1/Pms2 complex has an endonuclease activity suggested to play a role in the initiation of the excision step of MMR (24, 25). Downstream of mismatch recognition is a mispair excision step that can be catalyzed by Exonuclease 1 (Exo1) (26–28); however, defects in both S. cerevisiae and mouse Exo1 result in only a partial MMR deficiency, suggesting the existence of additional excision mechanisms (26, 27, 29). DNA polymerase δ, the single-strand DNA binding protein replication protein A (RPA), the sliding clamp proliferating cell nuclear antigen (PCNA), and the clamp loader replication factor C (RFC) are also required for MMR at different steps, including activation of Mlh1–Pms1/Pms2, stimulation of Exo1, potentially in Exo1-independent mispair excision, and in the gap-filling resynthesis steps of MMR (3, 16, 17, 24, 27, 30–36). Although much is known about these core MMR proteins, it is not well understood how eukaryotic MMR is coupled to DNA replication (1, 2), how excision is targeted to the newly replicated strand (1, 25, 37–39), or how different MMR mechanisms such as Exo1-dependent and -independent subpathways are selected or how many such subpathways exist (1, 24, 27, 29).S. cerevisiae has provided a number of tools for studying MMR, including forward genetic screens for mutations affecting MMR, including dominant and separation-of-function mutations, the ability to evaluate structure-based mutations in vivo, cell biological tools for visualizing and analyzing MMR proteins in vivo, and overproduction of individual MMR proteins for biochemical analysis. However, linking these tools with biochemical systems that catalyze MMR reactions in vitro for mechanistic studies has not yet been possible. Here, we describe the development of MMR reactions reconstituted using purified proteins for the analysis of MMR mechanisms.     

Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C₁₆H₁₅NO₄ (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.Toll-like receptors (TLRs) are type I transmembrane receptors that detect conserved “pathogen-associated molecular patterns” from microbes, as well as host-derived “danger-associated molecular patterns” (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4–9).Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (i.e., nuclear translocation of NF-κB, activation of MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in numerous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, ischemia/reperfusion injury) (11–14). Several inhibitors of TLR2 signaling have been developed (15–18), yet none is licensed for human use. A better understanding of the Toll/IL-1 receptor resistance (TIR) domain interactions involved in TLR2 signaling could lead to novel therapeutic agents.Both TLRs and adapter proteins contain a cytoplasmic TIR domain that mediates homotypic and heterotypic interactions during TLR signaling (19). Two adapter proteins implicated in TLR2 signaling are MyD88 and TIRAP (Mal). A conserved Pro [e.g., P681 in human TLR2 (hTLR2), P712 in murine TLR4 (mTLR4), P674 in hTLR10, P804 in mTLR11] within the BB loop of almost all TIR domains is critical for signaling (20–27). More importantly, the BB loop P681H mutation in hTLR2 abolished recruitment of MyD88 and signaling (20, 26). Based on this evidence, the BB loop within the TLR2 TIR domain appears to be an ideal target for attenuation of TLR2 signaling.Visual inspection of the crystal structure of the hTLR2 TIR domain (26) revealed a pocket formed by residues on the β-B strand and α-B helix that includes the highly conserved Pro and Gly residues of the BB loop. We hypothesized that targeting this pocket with a small molecule might inhibit interaction of TLR2 with MyD88, and thereby blunt TLR2 signaling. We identified C₁₆H₁₅NO₄ (C29) and its derivative, ortho-vanillin (o-vanillin), which inhibit mTLR2 and hTLR2 signaling initiated by synthetic and bacterial agonists without cytotoxicity. Interestingly, mutation of the BB loop pocket residues revealed a differential requirement for TLR2/1 vs. TLR2/6 signaling. Our data indicate that computer-aided drug design (CADD) is an effective approach for identifying small molecule inhibitors of TLR2 signaling and has the potential to identify inhibitors for other TLR signaling pathways.     

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ

Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia–neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K⁺ concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30–100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.Microglia are tissue-resident macrophages in the CNS that become activated in most brain disorders, such as bacterial meningoencephalitis, multiple sclerosis, and Alzheimer’s disease (1, 2). Activation of microglia features changes in morphology and receptor expression, antigen presentation, cytokine release, migration, and phagocytosis, and it ranges from proinflammatory and potentially neurotoxic to anti-inflammatory and neuroprotective phenotypes (1, 3, 4). The mechanisms that control the transition of microglia to reactive phenotypes, including the impact on neuronal function, are mostly unknown, however (5–7).Sensing of microbial or modified endogenous ligands by microglia is mediated by innate pattern recognition receptors, such as scavenger receptors and Toll-like receptors (TLRs). A prime example is TLR4, which acts with CD14, MD-2, and lipopolysaccharide (LPS)-binding protein in recognizing LPS, a cell wall component of Gram-negative bacteria (8, 9). TLR4 is also central to microglial recognition of amyloid-β peptide, which is thought to be part of the inflammatory response in Alzheimer’s disease (7, 10).LPS has been widely used to study the molecular mechanisms of microglial activation in inflammatory neurodegeneration (1–3). In primary monocultures and microglia-neuron cultures, LPS exposure alone or in combination with IFN-γ for a “booster” triggers the massive release of proinflammatory and cytotoxic factors, such as TNF-α, IL-6, and nitric oxide (NO), finally resulting in neuronal death (8, 11–18). Similar effects were observed in vivo after intracerebral administration of LPS (19–21). These and other studies have contributed to the concept that microglial TLR4 activation with LPS (i.e., with a single pathogenic stimulus) is sufficient to induce neurodegeneration (22, 23); however, this concept is biologically risky, and has been questioned in some experimental works and reviews (2–4, 11, 24, 25).Most previous studies focused on two aspects of microglial TLR4 activation with LPS: (i) the properties of the reactive microglial phenotype(s) and (ii) the degree of neurodegeneration. For this purpose, either simple culture systems or in vivo models, in which interactions with leukocytes infiltrating from the blood are inevitable, have been used (1, 4). Thus, it is widely unknown how TLR4 and IFN-γ receptor signaling in microglia individually contribute to neurotoxicity and neurodegeneration in situ. This aspect is highly relevant for several neurologic and psychiatric disorders. Moreover, concomitant alterations in neuronal information processing (i.e., dysfunction in excitatory pyramidal cells and inhibitory GABAergic interneurons, including astrocytes) have been little explored (25–27).We rigorously addressed these fundamental questions in postnatal neuronal tissue (1, 4). To mimic microglial confrontation with LPS in situ and, notably, in the absence of infiltrating leukocytes, we used organotypic hippocampal slice cultures that feature highly preserved cytoarchitectures and complex neuronal network functions (5, 28). Microglial interaction with infiltrating T helper type 1 (Th1) cells and/or natural killer (NK) cells was mimicked by recombinant IFN-γ administration.     

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56^bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α₄β₁ integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4⁺ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4⁺ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4⁺ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4⁺ T cells and the cytolytic activity of NK cells.Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease of the central nervous system (CNS) (1) and one of the major causes of neurological disability in young adults (2). MS is considered to be a primarily antigen-driven T cell-mediated disease with a complex genetic background influenced by environmental factors (1, 3) that is caused by an imbalanced immune-regulatory network (4). Among other well-known players of this network such as regulatory T cells and tolerogenic dendritic cells (DCs) (1), natural killer (NK) cells have been recently identified as additional factors in controlling homeostasis of antigen-activated T cells (5, 6).Originally discovered as antigen receptor-negative innate lymphocytes that play an important role in controlling virus-infected and tumor cells (7), NK cells have also been shown to suppress activated T cells through secretion of anti-inflammatory cytokines and/or cytolytic function (5, 6, 8–12). NK cells lyse target cells in a complex process depending on cell surface expression of certain inhibitory and activating receptors on NK cells and the corresponding ligands on target cells (13). Several activating NK-cell receptors–in particular, NKG2D (CD314) (5, 8, 9, 11, 14), the receptor for MIC-A/B and ULBP1-6, and DNAM-1 (DNAX accessory molecule, CD226) (6, 12, 15), the receptor for Nectin-2 (CD112) and poliovirus receptor (PVR/CD155)−have been proposed to be involved in NK cell-mediated lysis of activated T cells. Of note, polymorphisms in the gene encoding for DNAM-1 have been consistently found in MS-association studies (16–18). Both major NK-cell subsets, namely the CD56^brightCD16^dim/− and the CD56^dimCD16⁺ subsets (here referred to as CD56^bright and CD56^dim, respectively), seem to be capable of killing activated T cells (19). CD56^dim NK cells are the major NK-cell subset in the peripheral blood (PB) (90% of NK cells) and kill target cells without prior sensitization but only secrete low levels of cytokines (7, 20, 21), whereas CD56^bright NK cells are more abundant in secondary lymphoid tissues and inflammatory lesions (75–95% of NK cells), where they produce high amounts of immune-modulating cytokines but acquire cytolytic functions only after prolonged activation (7, 20, 21).Immune-modulating therapies targeting NK-cell frequencies and cytolytic functions among others such as IFN-β (22–24), glatiramer acetate (25), natalizumab (26, 27), fingolimod (28, 29), and daclizumab (10, 30, 31) point to an immune-protective role of both NK-cell subsets in MS. Daclizumab, a humanized antibody directed against the IL-2 receptor (IL-2R) α-chain (CD25) (reviewed in ref. 4) is a promising MS therapy, which recently showed superior efficacy compared with IFN-β in a phase III study (32). Expansion of peripheral (10, 33) as well as intrathecal (34) CD56^bright NK cells under daclizumab treatment correlated positively with therapeutic response (10, 30, 35). Nevertheless, it still remains to be elucidated whether NK-cell immune-regulatory functions are impaired as part of the disease process and whether modulation of the IL-2R with daclizumab restores these deficits or simply boosts NK-cell activity (4). Furthermore, the distribution and function of NK cells in active MS lesions is still poorly understood. Resolving the molecular basis of NK cell-mediated immune control and its potential impairment in MS is important for a better understanding of the role of NK cells in MS pathogenesis and the mechanism of action of NK cell-modulating therapies.The aim of the current study was to characterize the role of NK cells in the pathogenesis of MS by investigating the presence, distribution, and function of NK cells in three different compartments [CNS, cerebrospinal fluid (CSF), and PB]. Furthermore, a potential deficit in NK-cell immune-regulatory function, its underlying molecular mechanism, and the impact of IL-2R modulation by daclizumab high-yield process (DAC HYP) were explored by studying PB mononuclear cells (PBMCs) derived from clinically stable therapy-naïve MS patients and MS patients receiving daclizumab treatment in comparison with those derived from healthy individuals.     

Structure of NKp65 bound to its keratinocyte ligand reveals basis for genetically linked recognition in natural killer gene complex

The natural killer (NK) gene complex (NKC) encodes numerous C-type lectin-like receptors that govern the activity of NK cells. Although some of these receptors (Ly49s, NKG2D, CD94/NKG2A) recognize MHC or MHC-like molecules, others (Nkrp1, NKRP1A, NKp80, NKp65) instead bind C-type lectin-like ligands to which they are genetically linked in the NKC. To understand the basis for this recognition, we determined the structure of human NKp65, an activating receptor implicated in the immunosurveillance of skin, bound to its NKC-encoded ligand keratinocyte-associated C-type lectin (KACL). Whereas KACL forms a homodimer resembling other C-type lectin-like dimers, NKp65 is monomeric. The binding mode in the NKp65–KACL complex, in which a monomeric receptor engages a dimeric ligand, is completely distinct from those used by Ly49s, NKG2D, or CD94/NKG2A. The structure explains the exceptionally high affinity of the NKp65–KACL interaction compared with other cell–cell interaction pairs (K_D = 6.7 × 10⁻¹⁰ M), which may compensate for the monomeric nature of NKp65 to achieve cell activation. This previously unreported structure of an NKC-encoded receptor–ligand complex, coupled with mutational analysis of the interface, establishes a docking template that is directly applicable to other genetically linked pairs in the NKC, including Nkrp1–Clr, NKRP1A–LLT1, and NKp80–AICL.Natural killer (NK) cells are a fundamental component of innate immunity against tumors and virally infected cells. The cytolytic activity of NK cells is regulated by a dynamic interplay between activating and inhibitory signals transmitted by distinct classes of receptors that recognize both MHC and non-MHC ligands on the surface of target cells (1–3). In humans, these receptors are encoded in two distinct genomic regions: the leukocyte receptor complex (LRC) on chromosome 19 (4) and the NK gene complex (NKC) on chromosome 12 (5). The LRC codes for receptors belonging to the Ig superfamily. These include killer Ig-like receptors (KIRs), leukocyte Ig-like receptors, and the natural cytotoxicity receptor NKp46. The NKC codes for ∼30 cell-surface glycoproteins belonging to the C-type lectin-like superfamily (6). These receptors are expressed on NK and other immune-related cells, whose activity they regulate in various ways depending on cellular environment.NKC genes have been subdivided into killer cell lectin-like receptor (KLR) genes and C-type lectin receptor (CLEC) genes (6). KLR genes encode molecules expressed on NK cells, whereas CLEC genes encode molecules expressed on other cell types (e.g., CLEC2B and CLEC9A are expressed on myeloid and dendritic cells, respectively). The KLR family includes NKG2D and CD94/NKG2A (human and rodent) and rodent Ly49s. These receptors bind classical MHC class I (MHC-I) molecules or their structural relatives and thereby facilitate detection of stressed cells or cells exhibiting aberrant MHC-I expression (5).In addition, the KLR family includes receptors that do not engage ligands with an MHC-like fold, but instead interact with CLEC2 glycoproteins that are also members of the C-type lectin-like superfamily. These KLR and CLEC2 molecules, whose genes are intermingled in the telomeric subregion of the NKC, function as genetically linked receptor–ligand pairs. In mice, for example, the activating KLR family receptor Nkrp1f binds the CLEC2 family member Clrg, whereas the inhibitory receptor Nkrp1d binds Clrb (7, 8). Tumorigenesis and genotoxic stress down-regulate Clrb expression and thus promote NK cell-mediated lysis (8, 9). Corresponding Nkrp1–Clr receptor–ligand pairs have also been identified in humans. Thus, the inhibitory NK receptor NKRP1A (CD161), the human homolog of mouse Nkrp1d, engages the CLEC2 family member LLT1, which is expressed by activated dendritic and B cells, thereby negatively modulating NK-cell-mediated cytotoxicity (10–13). Another CLEC2 family member, AICL, is recognized by the activating NK receptor NKp80, which is genetically linked to AICL in the human NKC (14). Whereas NKp80 is found exclusively on NK cells, AICL is expressed on monocytes. The NKp80–AICL interaction promotes NK cell-mediated cytolysis of malignant myeloid cells and also mediates cellular cross-talk between NK cells and monocytes (14).The most recent addition to the human CLEC2 family is keratinocyte-associated C-type lectin (KACL or CLEC2A), whose expression is almost exclusively restricted to the skin, in marked contrast to the broad expression of other CLEC2 family members in hematopoietic cells (15). The receptor for KACL is NKp65, a distant relative of NKp80, which is encoded adjacent to KACL in the NKC in a tail-to-tail orientation (16). Similarly to NKp80 and AICL, no related sequences for NKp65 and KACL are present in rodents, although homologs of NKp80 and KACL exist in chimpanzee, rhesus macaque, and cow (15, 17). NKp65 stimulates NK cytotoxicity and release of proinflammatory cytokines upon engagement of ectopic KACL or of KACL on freshly isolated keratinocytes. The amino terminus of the cytoplasmic domain of NKp65 contains a hemi-ITAM motif that is required for NKp65-mediated cytotoxicity (16). This Syk kinase-recruiting motif is also found in other NKC-encoded activating receptors, including dectin-1, Clec1b, and NKp80 (17–19). The genetically linked NKp65–KACL receptor–ligand pair may fulfill a dedicated role in the immune surveillance of human skin through specific recognition of keratinocytes (16, 17).Considerable progress has been made in the structural analysis of NKC-encoded C-type lectin-like receptors that recognize MHC or MHC-related ligands (20). These structures include Ly49A bound to H-2D^d (21), Ly49C bound to H-2K^b (22, 23), NKG2D in complex with MICA (24), and NKG2A/CD94 in complex with HLA-E (25, 26). In addition, we determined the structure of killer cell lectin-like receptor G1 (KLRG1) bound to E-cadherin, a non-MHC ligand that is down-regulated in metastatic tumors (27). By contrast, no structural information is available for any of the NKC-encoded receptor–ligand pairs identified to date (Nkrp1f–Clrg and Nkrp1d–Clrb in rodents and NKRP1A–LLT1, NKp80–AICL, and NKp65–KACL in humans), except for the structures of mouse Nkrp1a and Clrg in unbound form (28, 29). To understand genetically linked recognition by C-type lectin-like receptors in the NKC at the atomic level, we determined the structure of NKp65 in complex with its keratinocyte ligand KACL.     

Autoinhibition and relief mechanism by the proteolytic processing of Toll-like receptor 8

Toll-like receptor 8 (TLR8) senses single-stranded RNA (ssRNA) and initiates innate immune responses. TLR8 requires proteolytic cleavage at the loop region (Z-loop) between leucine-rich repeat (LRR) 14 and LRR15 for its activation. However, the molecular basis of Z-loop processing remains unknown. To elucidate the mechanism of Z-loop processing, we performed biochemical and structural studies of how the Z-loop affects the function of TLR8. TLR8 with the uncleaved Z-loop is unable to form a dimer, which is essential for activation, irrespective of the presence of agonistic ligands. Crystallographic analysis revealed that the uncleaved Z-loop located on the ascending lateral face prevents the approach of the dimerization partner by steric hindrance. This autoinhibition mechanism of dimerization by the Z-loop might be occurring in the proteins of the same subfamily, TLR7 and TLR9.Toll-like receptors (TLRs) constitute a family of innate immune receptors that recognize pathogen-associated molecular patterns (1). The TLR molecule is a type I transmembrane protein characterized by an extracellular leucine-rich repeat (LRR) domain, a transmembrane helix, and an intracellular Toll/interleukin-1 receptor (TIR) homology domain (2). The typical TLR molecule is considered to be monomeric in the absence of ligands, transforming into an activated dimer form on ligand binding, which allows for dimerization of the intracellular TIR domain and subsequent signaling (2).The TLR subfamily comprising TLR7, TLR8, and TLR9 recognizes single-stranded (ss) nucleic acids from viruses and bacteria (3). Specifically, TLR7 and TLR8 recognize uridine- and guanosine-rich single-stranded RNA (ssRNA) (4–11), whereas TLR9 recognizes ssDNA containing the unmethylated cytosine-phosphate-guanine (CpG) dideoxynucleotide motif (12). Furthermore, TLR7 and TLR8 are also activated by synthetic chemical compounds (13, 14), such as imiquimod (TLR7-specific), resiquimod (R848; both TLR7 and TLR8), and CL075 (both TLR7 and TLR8).Certain regulation mechanisms of the functions of the TLR7–9 subfamily members are shared because of a high degree of sequence similarities (3). They reside on the endosomal membrane, and their transportation from endoplasmic reticulum (ER) to endolysosomes is mediated by the ER membrane protein Unc93B1 (15). Moreover, TLR7–9 possess a long inserted loop region (Z-loop), consisting of ∼30 amino acid residues, between LRR14 and LRR15, and the processing by proteolytic cleavage at the Z-loop is believed to be indispensable for their function (16–21). Specifically, the processing at the Z-loop of human TLR8 mediated by furin-like proprotein convertase and cathepsins produces functional TLR8 capable of ligand binding and signaling in endolysosomes. In addition, the cleaved form of TLR8 has been found to be predominant in immune cells (16). Recent structural studies demonstrate that the N- and C-terminal halves of TLR8 after Z-loop cleavage associate with each other, and that both fragments are cooperatively involved in ligand binding (22). Moreover, a recent study revealed that the latter half of the cleaved Z-loop interacts with LRRs to stabilize the TLR8 structure and contributes to ssRNA recognition by TLR8 (23).Although accumulating evidence illustrates the functional importance of Z-loop processing at the cellular level, mechanistic insights into this processing in the regulation of TLR8 function at the molecular level are lacking. Here, to unveil the mechanistic role of Z-loop processing of TLR8, we present the results of a combined structural and biochemical investigation of TLR8 with the uncleaved Z-loop.     

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3)

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2–glycans with the conserved Lewis^X binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam₂CSK₄ effectively, yet allows SSL3 to bind to an already formed TLR2–Pam₂CSK₄ complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2–lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.In recent years, Staphylococcus aureus has become a major health threat to both humans and domestic animals. It is found as a commensal bacterium in ∼30% of the human population, but when it becomes infectious it can cause a wide diversity of diseases, ranging from mild skin infections to life-threatening invasive conditions such as pneumonia and sepsis (1). Increased antibiotic resistance and a high amount of virulence factors secreted by S. aureus contribute to its emergence as a pathogen. Among these secreted virulence factors are the staphylococcal superantigen-like proteins (SSLs), a family of 14 proteins located on two genomic clusters (2–4). Recently, we and others identified SSL3 as a potent inhibitor of Toll-like receptor 2 (TLR2) (5, 6), an innate immunity receptor that is a dominant factor in immune recognition of S. aureus (7–10).TLR2 belongs to a family of 10 homologous innate immunity receptors that are activated by pathogen-associated molecular patterns (PAMPs) (11). TLR2 binds bacterial lipopeptides and lipoproteins. Subsequent formation of heterodimers with TLR1 or TLR6 leads to MyD88-dependent activation of the NF-κB pathway (12). TLR2 has dual ligand specificity that is determined by its dimerization partner; stimulation by diacyl lipopeptides from Gram-positive bacteria, including S. aureus, induces the formation of heterodimers with TLR6 (13), whereas triacyl lipopeptides from Gram-negative bacteria initiate formation of TLR2–TLR1 dimers (14). The structural basis for lipopeptide specificity was revealed by crystal structures of TLR2–TLR1 and TLR2–TLR6 complexes with their respective lipopeptide analogs Pam₃CSK₄ and Pam₂CSK₄: TLR2 binds two lipid tails in a large hydrophobic pocket, whereas the third lipid tail of triacyl lipopeptides is accommodated by a smaller pocket present in TLR1, but not in TLR6 (15, 16).The family of SSL proteins, including SSL3, share structural similarities to superantigens, but lack superantigenic activity. Interestingly, the functions that have been discovered for SSLs so far have all been linked to immune evasion. SSL5 inhibits neutrophil extravasation (17, 18) and phagocyte function (19, 20), SSL7 binds IgA and inhibits complement (21), and SSL10 inhibits IgG1-mediated phagocytosis (22, 23), blood coagulation (24), and the chemokine receptor CXCR4 (25). In addition to SSL3, also weak TLR2 inhibitory activity was observed for SSL4 (5), but it remains unknown whether that is its dominant function. This variety of immunomodulatory molecules and functions reflects the importance of the different components of our innate immune system in the defense against S. aureus (26).In this study we determined the crystal structures of SSL3 and the SSL3–TLR2 complex. In combination with mutagenesis and binding studies, our data provide a novel working mechanism of a functional TLR2 antagonist.     

Modeling putative therapeutic implications of exosome exchange between tumor and immune cells

Development of effective strategies to mobilize the immune system as a therapeutic modality in cancer necessitates a better understanding of the contribution of the tumor microenvironment to the complex interplay between cancer cells and the immune response. Recently, effort has been directed at unraveling the functional role of exosomes and their cargo of messengers in this interplay. Exosomes are small vesicles (30–200 nm) that mediate local and long-range communication through the horizontal transfer of information, such as combinations of proteins, mRNAs and microRNAs. Here, we develop a tractable theoretical framework to study the putative role of exosome-mediated cell–cell communication in the cancer–immunity interplay. We reduce the complex interplay into a generic model whose three components are cancer cells, dendritic cells (consisting of precursor, immature, and mature types), and killer cells (consisting of cytotoxic T cells, helper T cells, effector B cells, and natural killer cells). The framework also incorporates the effects of exosome exchange on enhancement/reduction of cell maturation, proliferation, apoptosis, immune recognition, and activation/inhibition. We reveal tristability—possible existence of three cancer states: a low cancer load with intermediate immune level state, an intermediate cancer load with high immune level state, and a high cancer load with low immune-level state, and establish the corresponding effective landscape for the cancer–immunity network. We illustrate how the framework can contribute to the design and assessments of combination therapies.Immunotherapeutic approaches have recently emerged as effective therapeutic modalities (1) exemplified by immune checkpoint blockade with anti–CTLA-4 to activate T-cells and induce tumor cell killing, which has been shown to be effective for some cancers but not others (2). A better understanding of the intricate interplay between cancer and the immune system, and of mechanisms of immune evasion and of hijacking of the host response by cancer cells, is relevant to the development of effective immunotherapeutic approaches (3–6).The immune-based suppression of tumor development and progression is mediated through nonspecific innate immunity and antigen-specific adaptive immunity (7). However, cancer cells can inhibit the immune response, thus evading suppression in multiple ways (8) (see below for details), and additionally hijack the immune system to their advantage (3, 4). The challenge to understand the tumor–immune interplay stems from the dynamic nature, and complexity and heterogeneity of both the cancer cells and the immune system and their interactions through the tumor microenvironment (9).Here we consider immune cells as consisting of macrophages (10), natural killer cells (11), cytotoxic T cells (12), helper T cells (13), and regulatory T cells (3). These various immune cells are produced, activated, and perform their functions separated by space and time, which contributes to additional complexity (14). Among the immune cells, dendritic cells (DCs) are the most efficient antigen-presenting cells to bridge innate immunity with adaptive immunity (15). DCs also secrete cytokines that promote the antitumor functions of both natural killer cells and macrophages (16, 17). We consider the tumor microenvironment as comprised of a heterogeneous population of cancer cells (18), stromal cells (19), and tumor-infiltrating immune cells (20). The interactions among these cell types contribute to tumor development and progression. Tumor-associated macrophages and cancer-associated fibroblasts regulate tumor metabolism and engender an immune-suppressive environment by secreting TGF-β and other cytokines (21). Fluctuations in energy sources and oxygen within a tumor contribute to malignant progression and cell phenotypic diversity (22, 23).Though secreted factors play critical roles in cell–cell communications, here we focus on the additional role of cell–cell communication mediated by the exchange of special extracellular lipid vesicles called exosomes (24). These nanovesicles of ∼30–200 nm are formed in the multivesicular bodies and then released from the cell into the extracellular space (25). The exosomes carry a broad range of cargo, including proteins, microRNAs, mRNAs, and DNA fragments, to specific target cells at a remote location (26). Membrane markers assign the exosomes to specific targeted cells. Notably, upon entering the target cell, the exosomes induce modulation of cell function and even identity switch (phenotypic, epigenetic, and even genetic) (27). Exosomes have recently emerged as playing an important role in the immune system interaction with tumors (28, 29). Tumor-derived exosomes can promote metastatic niche formation by influencing bone marrow-derived cells toward a prometastatic phenotype through upregulation of c-Met (29). DCs have been shown to induce tumor cell killing through release of exosomes that contain potent tumor-suppressive factors such as TNF and through activation of natural killer cells, cytotoxic T cells, and helper T cells (24, 30–32). However, tumor-derived exosomes (Tex) can directly inhibit the differentiation of DCs in bone marrow (33), which strongly inhibits the dendritic cell-mediated immune response to the tumor. In addition, Tex can also directly inhibit natural killer cells (34).Mathematical models have been devised to study the complex interactions of cancer and immune system, including those that consider spatial heterogeneity (as reviewed in ref. 35) and those that consider spatially homogeneous populations (as reviewed in ref. 36). Cancer–immunity models have been constructed to investigate the effects of therapy (37–39), cancer dormancy (40), and interactions with time delay (41). Other types of modeling methods have also been applied. For example, tumor growth has also been fitted to experimental data by artificial neural networks (42); a detailed network of cancer immune system has been modeled by multiple subset models (43).In this study, we have developed an exosome exchange-based cancer–immunity interplay (ECI) model, to incorporate the special role of DCs and exosome-mediated communications. Distinct from the previous approaches, our modeling strategy is adapted from methodology used in studies of gene regulatory circuits, allowing us to check the multistability features of the system (44). We find that, by including exosome exchange, the cancer–immunity interplay can give rise to three quasi-stable cancer states, which may be associated with the elimination/equilibrium/escape phases proposed in the immunoediting theory (45). The ECI model is also capable of explaining tumorigenesis by considering the time evolution of immune responses. Guided by the treatment simulations, we assess the effectiveness of various therapeutic protocols with and without time delay and noise.     

Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1–dependent NK cell-mediated lysis of infected cells

Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2–infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry.Alphaherpesviruses constitute the largest subfamily of the herpesviruses, comprising closely related and important pathogens like herpes simplex virus (HSV) in man, pseudorabies virus (PRV) in pigs, and bovine herpesvirus 1 (BHV-1) in cattle.Natural killer (NK) cells play a central role in the defense against viral infections and cancer development. Functional NK cells are of particular importance in preventing herpesviruses from causing aggravated disease, including life-threatening encephalitis for alphaherpesviruses like HSV and varicella-zoster virus (1–3). The significance of the NK cell response against herpesviruses is also reflected by the various mechanisms that these pathogens have evolved to evade or delay it (4). Indeed, for beta- and gammaherpesviruses, a variety of molecular mechanisms avoiding the NK-mediated antiviral activity have been defined (4–16). Remarkably and paradoxically, such mechanisms have remained largely elusive for the alphaherpesviruses (17).Identifying and understanding these mechanisms is of particular relevance for alphaherpesviruses also in view of the potential therapeutic applications of HSV. Indeed, a limiting factor in HSV vector-based oncotherapy is the premature clearance of the viral vector by NK cells (18).NK cell activity is regulated by an array of germline-encoded activating and inhibitory surface receptors capable of transducing signals upon engagement by their respective ligands (19, 20). The sum of these signals determines the outcome of NK cell effector responses including cytotoxicity against NK-sensitive targets (20). A variety of NK activating receptors are involved in recognition of virus-infected cells (18, 21–26). One of the important NK activating receptors is DNAX accessory molecule 1 (DNAM-1), which binds to CD112 (nectin-2) and CD155 (poliovirus receptor, PVR), whose expression can be induced in both virus infected and tumor cells (5, 26–28).Interestingly, the viral gD envelope glycoproteins of certain human and animal alphaherpesviruses, including HSV-2, PRV, and BHV-1, interact with CD112 and/or CD155 to facilitate viral entry (29, 30). HSV-1 gD does not typically display substantial affinity for CD112, except for particular HSV-1 isolates, including some retrieved from patients with encephalitis (31).In the current study, we demonstrate that the significance of these virus ligand-cellular receptor interactions can stretch beyond virus entry and can influence immune recognition. We report that expression of gD of PRV and HSV-2 reduces DNAM-1–mediated cell lysis by NK cells through suppression of CD112 levels in infected and transfected cells. The gD/CD112/DNAM-1 interplay identified here may have consequences for the development of medical applications ranging from vaccination to oncolytic virotherapy.     

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.Solid tumors represent a complex mass of tissue composed of multiple distinct cell types (1, 2). Cells within the tumor produce a range of soluble factors to create a complex of signaling networks within the tumor microenvironment (3–7). One of the outcomes of this crosstalk is tumor-promoting inflammation (TPI) (8, 9). TPI can modulate the functions of tumor-infiltrating myeloid lineage cells including macrophages (10–12). Tumor-associated macrophages (TAMs) consistently display an alternatively activated phenotype (M2) commonly found in sites of wound healing (13–18). These macrophages promote tumor growth while suppressing the host immune response locally (19–22). Polarization and subversion of tumor-infiltrating macrophages is accomplished via immune mediators in the tumor microenvironment (23, 24). Adding to the complexity of solid tumors is the heterogeneity of the cancer cells (2). Tumor cells of varying differentiation states and different characteristics coexist within a tumor (25–29). However, the different roles of each tumor cell subset during cancer progression remain undefined.Bladder cancer (BC) represents a growing number of solid tumors characterized by the infiltration of a significant number of myeloid cells in the neoplastic lesion (30, 31). We have previously determined that keratin 14 (KRT14) expression marks the most primitive differentiation state in BC cells (32). KRT14 expression is significantly associated with poor overall patient survival. However, the mechanisms used by KRT14-expressing cells to promote tumor growth remain unclear. In the current study, we found that KRT14+ basal BC cells also express higher levels of CD14. Here, we investigate the strategies used by KRT14+ CD14-high BC cells to promote tumor growth.     

Platelets guide the formation of early metastatic niches

During metastasis, host cells are recruited to disseminated tumor cells to form specialized microenvironments (“niches”) that promote metastatic progression, but the mechanisms guiding the assembly of these niches are largely unknown. Tumor cells may autonomously recruit host cells or, alternatively, host cell-to-host cell interactions may guide the formation of these prometastatic microenvironments. Here, we show that platelet-derived rather than tumor cell-derived signals are required for the rapid recruitment of granulocytes to tumor cells to form “early metastatic niches.” Granulocyte recruitment relies on the secretion of CXCL5 and CXCL7 chemokines by platelets upon contact with tumor cells. Blockade of the CXCL5/7 receptor CXCR2, or transient depletion of either platelets or granulocytes prevents the formation of early metastatic niches and significantly reduces metastatic seeding and progression. Thus, platelets recruit granulocytes and guide the formation of early metastatic niches, which are crucial for metastasis.Interactions between host cells and tumor cells both at the primary and metastatic sites are crucial for efficient metastasis (1–3). At the site of metastasis, host cell–tumor cell cross talk contributes to the formation of a metastatic niche, a specialized microenvironment necessary for disease progression. Leukocytes, in particular monocytes and macrophages, have been shown to be recruited to metastatic niches and to support metastasis (4–10). Mechanistically, macrophages secrete VEGFA that facilitates tumor cell extravasation (4). In addition, interaction of monocytes/macrophages with tumor cells via VCAM1 can prevent tumor cell apoptosis and allow reinitiation of growth at the metastatic site (8, 9). Granulocytes have also been reported to interact with tumor cells and facilitate metastatic seeding possibly by enhancing tumor cell arrest and extravasation (11–13). However, granulocytes exposed to specific environmental stimuli can kill metastatic tumor cells, suggesting that their role in metastasis is highly context dependent (14, 15).Platelets, which interact with tumor cells during their transit through the circulation, also enhance metastasis via multiple mechanisms (3, 16). Metastatic tumor cells can express high levels of tissue factor (TF) and adhesion molecules, such as P-selectin ligands, through which they bind to and activate platelets (16). These interactions result in the formation by platelets of a physical shield around tumor cells, which prevents attacks from natural killer (NK) cells and promotes tumor cell adhesion to the endothelium (17–19). In addition, platelets also promote tumor cell extravasation by increasing endothelial permeability and by directly signaling to tumor cells to enhance their invasive and metastatic potential (20, 21). In particular, direct contact between platelets and tumor cells triggers the TGFβ1 and NF-κB signaling pathways in tumor cells, which induce an epithelial–mesenchymal transition and promote metastasis (20). Given their important signaling roles, platelets may influence metastasis by signaling not only to tumor cells but also to host cells forming metastatic niches. Furthermore, whether platelets and leukocytes are independently recruited by cancer cells to the site of metastasis, or whether hierarchical interactions among host cells drive the formation of metastatic niches remains unknown. Here, we define the relative roles of platelets and leukocytes during the early stages of metastatic seeding and the subsequent impact of these interactions on metastatic progression.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.