Cytosolic bacterial pathogens activate TLR pathways in tumors that synergistically enhance STING agonist cancer therapies.

Bacterial pathogens that invade the eukaryotic cytosol are distinctive tools for fighting cancer, as they preferentially target tumors and can deliver cancer antigens to MHC-I. Cytosolic bacterial pathogens have undergone extensive preclinical development and human clinical trials, yet the molecular mechanisms by which they are detected by innate immunity in tumors is unclear. We report that intratumoral delivery of phylogenetically distinct cytosolic pathogens, including Listeria, Rickettsia, and Burkholderia species, elicited anti-tumor responses in established, poorly immunogenic melanoma and lymphoma in mice. We were surprised to observe that although the bacteria required entry to the cytosol, the anti-tumor responses were largely independent of the cytosolic sensors cGAS/STING and instead required TLR signaling. Combining pathogens with TLR agonists did not enhance anti-tumor efficacy, while combinations with STING agonists elicited profound, synergistic anti-tumor effects with complete responses in >80% of mice after a single dose. Small molecule TLR agonists also synergistically enhanced the anti-tumor activity of STING agonists. The anti-tumor effects were diminished in Rag2-deficient mice and upon CD8 T cell depletion. Mice cured from combination therapy developed immunity to cancer rechallenge that was superior to STING agonist monotherapy. Together, these data provide a framework for enhancing the efficacy of microbial cancer therapies and small molecule innate immune agonists, via the co-activation of STING and TLRs.

A patatin-like phospholipase mediates Rickettsia parkeri escape from host membranes.

Rickettsia species of the spotted fever group are arthropod-borne obligate intracellular bacteria that can cause mild to severe human disease. These bacteria invade host cells, replicate in the cell cytosol, and spread from cell to cell. To access the host cytosol and avoid immune detection, they escape membrane-bound vacuoles by expressing factors that disrupt host membranes. Here, we show that a patatin-like phospholipase A2 enzyme (Pat1) facilitates Rickettsia parkeri infection by promoting escape from host membranes and cell-cell spread. Pat1 is important for infection in a mouse model and, at the cellular level, is crucial for efficiently escaping from single and double membrane-bound vacuoles into the host cytosol, and for avoiding host galectins that mark damaged membranes. Pat1 is also important for avoiding host polyubiquitin, preventing recruitment of autophagy receptor p62, and promoting actin-based motility and cell-cell spread.

Interferon receptor-deficient mice are susceptible to eschar-associated rickettsiosis.

Arthropod-borne rickettsial pathogens cause mild and severe human disease worldwide. The tick-borne pathogen Rickettsia parkeri elicits skin lesions (eschars) and disseminated disease in humans; however, inbred mice are generally resistant to infection. We report that intradermal infection of mice lacking both interferon receptors (Ifnar1-/-;Ifngr1-/-) with as few as 10 R. parkeri elicits eschar formation and disseminated, lethal disease. Similar to human infection, eschars exhibited necrosis and inflammation, with bacteria primarily found in leukocytes. Using this model, we find that the actin-based motility factor Sca2 is required for dissemination from the skin to internal organs, and the outer membrane protein OmpB contributes to eschar formation. Immunizing Ifnar1-/-;Ifngr1-/- mice with sca2 and ompB mutant R. parkeri protects against rechallenge, revealing live-attenuated vaccine candidates. Thus, Ifnar1-/-;Ifngr1-/- mice are a tractable model to investigate rickettsiosis, virulence factors, and immunity. Our results further suggest that discrepancies between mouse and human susceptibility may be due to differences in interferon signaling.

Tick bites allow disease-causing microbes, including multiple species of Rickettsia bacteria, to pass from arthropods to humans. Being exposed to Rickettsia parkeri, for example, can cause a scab at the bite site, fever, headache and fatigue. To date, no vaccine is available against any of the severe diseases caused by Rickettsia species. Modelling human infections in animals could help to understand and combat these illnesses. R. parkeri is a good candidate for such studies, as it can give insight into more severe Rickettsia infections while being comparatively safer to handle. However, laboratory mice are resistant to this species of bacteria, limiting their use as models. To explore why this is the case, Burke et al. probed whether an immune mechanism known as interferon signalling protects laboratory rodents against R. parkeri. During infection, the immune system releases molecules called interferons that stick to 'receptors' at the surface of cells, triggering defense mechanisms that help to fight off an invader. Burke et al. injected R. parkeri into the skin of mice that had or lacked certain interferon receptors, showing that animals without two specific receptors developed scabs and saw the disease spread through their body. Further investigation showed that two R. parkeri proteins, known as OmpB or Sca2, were essential for the bacteria to cause skin lesions and damage internal organs. Burke et al. then used R. parkeri that lacked OmpB or Sca2 to test whether these modified, inoffensive microbes could act as 'vaccines'. And indeed, vulnerable laboratory mice which were first exposed to the mutant bacteria were then able to survive the 'normal' version of the microbe. Together, this work reveals that interferon signalling protects laboratory mice against R. parkeri infections. It also creates an animal model that can be used to study disease and vaccination.

Lysine methylation shields an intracellular pathogen from ubiquitylation and autophagy.

Many intracellular pathogens avoid detection by their host cells. However, it remains unknown how they avoid being tagged by ubiquitin, an initial step leading to antimicrobial autophagy. Here, we show that the intracellular bacterial pathogen Rickettsia parkeri uses two protein-lysine methyltransferases (PKMTs) to modify outer membrane proteins (OMPs) and prevent their ubiquitylation. Mutants deficient in the PKMTs were avirulent in mice and failed to grow in macrophages because of ubiquitylation and autophagic targeting. Lysine methylation protected the abundant surface protein OmpB from ubiquitin-dependent depletion from the bacterial surface. Analysis of the lysine-methylome revealed that PKMTs modify a subset of OMPs, including OmpB, by methylation at the same sites that are modified by host ubiquitin. These findings show that lysine methylation is an essential determinant of rickettsial pathogenesis that shields bacterial proteins from ubiquitylation to evade autophagic targeting.

Inflammasome-mediated antagonism of type I interferon enhances Rickettsia pathogenesis.

The innate immune system fights infection with inflammasomes and interferons. Facultative bacterial pathogens that inhabit the host cytosol avoid inflammasomes1-6 and are often insensitive to type I interferons (IFN-I), but are restricted by IFN-γ7-11. However, it remains unclear how obligate cytosolic bacterial pathogens, including Rickettsia species, interact with innate immunity. Here, we report that the human pathogen Rickettsia parkeri is sensitive to IFN-I and benefits from inflammasome-mediated host cell death that antagonizes IFN-I. R. parkeri-induced cell death requires the cytosolic lipopolysaccharide (LPS) receptor caspase-11 and antagonizes IFN-I production mediated by the DNA sensor cGAS. The restrictive effects of IFN-I require the interferon regulatory factor IRF5, which upregulates genes encoding guanylate-binding proteins (GBPs) and inducible nitric oxide synthase (iNOS), which we found to inhibit R. parkeri. Mice lacking both IFN-I and IFN-γ receptors succumb to R. parkeri, revealing critical and overlapping roles for these cytokines in vivo. The interactions of R. parkeri with inflammasomes and interferons are similar to those of viruses, which can exploit the inflammasome to avoid IFN-I12, are restricted by IFN-I via IRF513,14, and are controlled by IFN-I and IFN-γ in vivo15-17. Our results suggest that the innate immune response to an obligate cytosolic bacterial pathogen lies at the intersection of antibacterial and antiviral responses.

A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics.

Gram-negative bacteria in the order Rickettsiales have an obligate intracellular growth requirement, and some species cause human diseases such as typhus and spotted fever. The bacteria have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome reduction. However, it remains largely unknown which nutrients they acquire and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the Rickettsiales that has resulted from reductive genome evolution. Furthermore, we investigated whether the spotted fever group Rickettsia species Rickettsia parkeri scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry, we found that infection caused decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that treatment of infected cells with statins, which inhibit host isoprenoid synthesis, prohibited bacterial growth. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting that statins lead to an inhibition of cell wall synthesis. Altogether, our results describe a potential Achilles' heel of obligate intracellular pathogens that can potentially be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth.IMPORTANCE Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen Rickettsia parkeri on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles' heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.

Evasion of autophagy mediated by Rickettsia surface protein OmpB is critical for virulence.

Rickettsia are obligate intracellular bacteria that evade antimicrobial autophagy in the host cell cytosol by unknown mechanisms. Other cytosolic pathogens block different steps of autophagy targeting, including the initial step of polyubiquitin-coat formation. One mechanism of evasion is to mobilize actin to the bacterial surface. Here, we show that actin mobilization is insufficient to block autophagy recognition of the pathogen Rickettsia parkeri. Instead, R. parkeri employs outer membrane protein B (OmpB) to block ubiquitylation of the bacterial surface proteins, including OmpA, and subsequent recognition by autophagy receptors. OmpB is also required for the formation of a capsule-like layer. Although OmpB is dispensable for bacterial growth in endothelial cells, it is essential for R. parkeri to block autophagy in macrophages and to colonize mice because of its ability to promote autophagy evasion in immune cells. Our results indicate that OmpB acts as a protective shield to obstruct autophagy recognition, thereby revealing a distinctive bacterial mechanism to evade antimicrobial autophagy.

RECON-Dependent Inflammation in Hepatocytes Enhances Listeria monocytogenes Cell-to-Cell Spread.

The oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs). CDN binding inhibits RECON's enzymatic activity and subsequently promotes inflammation. In this study, we sought to characterize the effects of RECON on the infection cycle of the intracellular bacterium Listeria monocytogenes, which secretes cyclic di-AMP (c-di-AMP) into the cytosol of infected host cells. Here, we report that during infection of RECON-deficient hepatocytes, which exhibit hyperinflammatory responses, L. monocytogenes exhibits significantly enhanced cell-to-cell spread. Enhanced bacterial spread could not be attributed to alterations in PrfA or ActA, two virulence factors critical for intracellular motility and intercellular spread. Detailed microscopic analyses revealed that in the absence of RECON, L. monocytogenes actin tail lengths were significantly longer and there was a larger number of faster-moving bacteria. Complementation experiments demonstrated that the effects of RECON on L. monocytogenes spread and actin tail lengths were linked to its enzymatic activity. RECON enzyme activity suppresses NF-κB activation and is inhibited by c-di-AMP. Consistent with these previous findings, we found that augmented NF-κB activation in the absence of RECON caused enhanced L. monocytogenes cell-to-cell spread and that L. monocytogenes spread correlated with c-di-AMP secretion. Finally, we discovered that, remarkably, increased NF-κB-dependent inducible nitric oxide synthase expression and nitric oxide production were responsible for promoting L. monocytogenes cell-to-cell spread. The work presented here supports a model whereby L. monocytogenes secretion of c-di-AMP inhibits RECON's enzymatic activity, drives augmented NF-κB activation and nitric oxide production, and ultimately enhances intercellular spread.IMPORTANCE To date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement of L. monocytogenes cell-to-cell spread. This is a heretofore-unknown role of these molecules and suggests L. monocytogenes may benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, like L. monocytogenes, they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell.

The Unexpected Effects of the Combination of Antibiotics and Immunity.

ß-lactam antibiotics and immune enzymes, including lysozyme, kill bacteria by rupturing the cell wall. Curiously, their combination can select for viable, wall-less bacteria. Kawai and colleagues describe the molecular details regarding the emergence of these forms, illustrating a novel and potentially clinically relevant mechanism by which bacteria escape killing by antibiotics.

SpoVG Is a Conserved RNA-Binding Protein That Regulates Listeria monocytogenes Lysozyme Resistance, Virulence, and Swarming Motility.

UNLABELLED: In this study, we sought to characterize the targets of the abundant Listeria monocytogenes noncoding RNA Rli31, which is required for L. monocytogenes lysozyme resistance and pathogenesis. Whole-genome sequencing of lysozyme-resistant suppressor strains identified loss-of-expression mutations in the promoter of spoVG, and deletion of spoVG rescued lysozyme sensitivity and attenuation in vivo of the rli31 mutant. SpoVG was demonstrated to be an RNA-binding protein that interacted with Rli31 in vitro. The relationship between Rli31 and SpoVG is multifaceted, as both the spoVG-encoded protein and the spoVG 5'-untranslated region interacted with Rli31. In addition, we observed that spoVG-deficient bacteria were nonmotile in soft agar and suppressor mutations that restored swarming motility were identified in the gene encoding a major RNase in Gram-positive bacteria, RNase J1. Collectively, these findings suggest that SpoVG is similar to global posttranscriptional regulators, a class of RNA-binding proteins that interact with noncoding RNA, regulate genes in concert with RNases, and control pleiotropic aspects of bacterial physiology. IMPORTANCE: spoVG is widely conserved among bacteria; however, the function of this gene has remained unclear since its initial characterization in 1977. Mutation of spoVG impacts various phenotypes in Gram-positive bacteria, including methicillin resistance, capsule formation, and enzyme secretion in Staphylococcus aureus and also asymmetric cell division, hemolysin production, and sporulation in Bacillus subtilis. Here, we demonstrate that spoVG mutant strains of Listeria monocytogenes are hyper-lysozyme resistant, hypervirulent, nonmotile, and misregulate genes controlling carbon metabolism. Furthermore, we demonstrate that SpoVG is an RNA-binding protein. These findings suggest that SpoVG has a role in L. monocytogenes, and perhaps in other bacteria, as a global gene regulator. Posttranscriptional gene regulators help bacteria adapt to various environments and coordinate differing aspects of bacterial physiology. SpoVG may help the organism coordinate environmental growth and virulence to survive as a facultative pathogen.

Quantitative Analysis of Technological Innovation in Knee Arthroplasty: Using Patent and Publication Metrics to Identify Developments and Trends.

BACKGROUND: Surgery is in a constant continuum of innovation with refinement of technique and instrumentation. Arthroplasty surgery potentially represents an area with highly innovative process. This study highlights key area of innovation in knee arthroplasty over the past 35 years using patent and publication metrics. Growth rates and patterns are analyzed. Patents are correlated to publications as a measure of scientific support. METHODS: Electronic patent and publication databases were searched over the interval 1980-2014 for "knee arthroplasty" OR "knee replacement." The resulting patent codes were allocated into technology clusters. Citation analysis was performed to identify any important developments missed on initial analysis. The technology clusters identified were further analyzed, individual repeat searches performed, and growth curves plotted. RESULTS: The initial search revealed 3574 patents and 16,552 publications. The largest technology clusters identified were Unicompartmental, Patient-Specific Instrumentation (PSI), Navigation, and Robotic knee arthroplasties. The growth in patent activity correlated strongly with publication activity (Pearson correlation value 0.892, P < .01), but was growing at a faster rate suggesting a decline in vigilance. PSI, objectively the fastest growing technology in the last 5 years, is currently in a period of exponential growth that began a decade ago. Established technologies in the study have double s-shaped patent curves. CONCLUSION: Identifying trends in emerging technologies is possible using patent metrics and is useful information for training and regulatory bodies. The decline in ratio of publications to patents and the uninterrupted growth of PSI are developments that may warrant further investigation.

A prl mutation in SecY suppresses secretion and virulence defects of Listeria monocytogenes secA2 mutants.

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.

Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.

Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection.

Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-ß) and coregulated genes. We previously identified the IFN-ß stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-ß. One mutant that stimulated elevated levels of IFN-ß harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-ß expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.

Activation of a plant nucleotide binding-leucine rich repeat disease resistance protein by a modified self protein.

Nucleotide binding-leucine rich repeat (NB-LRR) proteins function as intracellular receptors for the detection of pathogens in both plants and animals. Despite their central role in innate immunity, the molecular mechanisms that govern NB-LRR activation are poorly understood. The Arabidopsis NB-LRR protein RPS5 detects the presence of the Pseudomonas syringae effector protein AvrPphB by monitoring the status of the Arabidopsis protein kinase PBS1. AvrPphB is a cysteine protease that targets PBS1 for cleavage at a single site within the activation loop of PBS1. Using a transient expression system in the plant Nicotiana benthamiana and stable transgenic Arabidopsis plants we found that both PBS1 cleavage products are required to activate RPS5 and can do so in the absence of AvrPphB. We also found, however, that the requirement for cleavage of PBS1 could be bypassed simply by inserting five amino acids at the PBS1 cleavage site, which is located at the apex of the activation loop of PBS1. Activation of RPS5 did not require PBS1 kinase function, and thus RPS5 appears to sense a subtle conformational change in PBS1, rather than cleavage. This finding suggests that NB-LRR proteins may function as fine-tuned sensors of alterations in the structures of effector targets.

Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity.

Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1ß and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes, strains that ectopically secreted flagellin induced robust host cell death and IL-1ß secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes. Attenuation in vivo was dependent on Nlrc4, but independent of IL-1ß/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.