Farnesol remodels the peritoneal cavity immune environment influencing Candida albicans pathogenesis during intra-abdominal infection.

Candida albicans is a lifelong member of the mycobiome causing mucosal candidiasis and life-threatening, systemic, and intra-abdominal disease in immunocompromised and transplant patients. Despite the clinical importance of intra-abdominal candidiasis with mortality rates between 40% and 70%, the contribution of fungal virulence factors and host immune responses to disease has not been extensively studied. Secretion of the quorum-sensing molecule, farnesol, acts as a virulence factor for C. albicans during systemic infection, while inducing local, protective innate immune responses in oral models of infection. Previously, we reported that farnesol recruits macrophages to the peritoneal cavity in mice, suggesting a role for farnesol in innate immune responses. Here, we expand on our initial findings, showing that farnesol profoundly alters the peritoneal cavity microenvironment promoting innate inflammation. Intra-peritoneal injection of farnesol stimulates rapid local death of resident peritoneal cells followed by recruitment of neutrophils and inflammatory macrophages into the peritoneal cavity and peritoneal mesothelium associated with an early increase in chemokines followed by proinflammatory cytokines. These rapid inflammatory responses to farnesol significantly increase morbidity and mortality of mice with intra-abdominal candidiasis associated with increased formation of peritoneal adhesions, despite similar rates of fungal clearance from the peritoneal cavity and retro-peritoneal organs. C. albicans ddp3Δ/ddp3Δ knockout and reconstituted strains recapitulate these findings. This indicates that farnesol may be detrimental to the host during intra-abdominal infections. Importantly, our results highlight a need to understand how C. albicans virulence factors modulate the host immune response within the peritoneum, an exceedingly common site of Candida infection.

Influence of two-dimensional expiratory airflow variations on respiratory particle propagation during pronunciation of the fricative [f].

Propagation of respiratory particles, potentially containing viable viruses, plays a significant role in the transmission of respiratory diseases (e.g., COVID-19) from infected people. Particles are produced in the upper respiratory system and exit the mouth during expiratory events such as sneezing, coughing, talking, and singing. The importance of considering speaking and singing as vectors of particle transmission has been recognized by researchers. Recently, in a companion paper, dynamics of expiratory flow during fricative utterances were explored, and significant variations of airflow jet trajectories were reported. This study focuses on respiratory particle propagation during fricative productions and the effect of airflow variations on particle transport and dispersion as a function of particle size. The commercial ANSYS-Fluent computational fluid dynamics (CFD) software was employed to quantify the fluid flow and particle dispersion from a two-dimensional mouth model of sustained fricative [f] utterance as well as a horizontal jet flow model. The fluid velocity field and particle distributions estimated from the mouth model were compared with those of the horizontal jet flow model. The significant effects of the airflow jet trajectory variations on the pattern of particle transport and dispersion during fricative utterances were studied. Distinct differences between the estimations of the horizontal jet model for particle propagation with those of the mouth model were observed. The importance of considering the vocal tract geometry and the failure of a horizontal jet model to properly estimate the expiratory airflow and respiratory particle propagation during the production of fricative utterances were emphasized.

Modeling an immune response to influenza A virus infection in alveolar epithelial cells.

Influenza A viruses (IAV) have been the cause of several influenza pandemics in history and are a significant threat for the next global pandemic. Hospitalized influenza patients often have excess interferon production and a dysregulated immune response to the IAV infection. Obtaining a better understanding of the mechanisms of IAV infection that induce these harmful effects would help drug developers and health professionals create more effective treatments for IAV infection and improve patient outcomes. IAV stimulates viral sensors and receptors expressed by alveolar epithelial cells, like RIG-I and toll-like receptor 3 (TLR3). These two pathways coordinate with one another to induce expression of type III interferons to combat the infection. Presented here is a queuing theory-based model of these pathways that was designed to analyze the timing and amount of interferons produced in response to IAV single stranded RNA and double-stranded RNA detection. The model accurately represents biological data showing the necessary coordination of the RIG-I and TLR3 pathways for effective interferon production. This model can serve as the framework for future studies of IAV infection and identify new targets for potential treatments.

Variability in expiratory trajectory angles during consonant production by one human subject and from a physical mouth model: Application to respiratory droplet emission.

The COVID-19 pandemic has highlighted the need to improve understanding of droplet transport during expiratory emissions. While historical emphasis has been placed on violent events such as coughing and sneezing, the recognition of asymptomatic and presymptomatic spread has identified the need to consider other modalities, such as speaking. Accurate prediction of infection risk produced by speaking requires knowledge of both the droplet size distributions that are produced, as well as the expiratory flow fields that transport the droplets into the surroundings. This work demonstrates that the expiratory flow field produced by consonant productions is highly unsteady, exhibiting extremely broad inter- and intra-consonant variability, with mean ejection angles varying from ≈+30° to -30°. Furthermore, implementation of a physical mouth model to quantify the expiratory flow fields for fricative pronunciation of [f] and [θ] demonstrates that flow velocities at the lips are higher than previously predicted, reaching 20-30 m/s, and that the resultant trajectories are unstable. Because both large and small droplet transport are directly influenced by the magnitude and trajectory of the expirated air stream, these findings indicate that prior investigations of the flow dynamics during speech have largely underestimated the fluid penetration distances that can be achieved for particular consonant utterances.

High- and low-molecular-weight chitosan act as adjuvants during single-dose influenza A virus protein vaccination through distinct mechanisms.

The investigation of new adjuvants is essential for the development of efficacious vaccines. Chitosan (CS), a derivative of chitin, has been shown to act as an adjuvant, improving vaccine-induced immune responses. However, the effect of CS molecular weight (MW) on this adjuvanticity has not been investigated, despite MW having been shown to impact CS biological properties. Here, two MW variants of CS were investigated for their ability to enhance vaccine-elicited immune responses in vitro and in vivo, using a single-dose influenza A virus (IAV) protein vaccine model. Both low-molecular-weight (LMW) and high-molecular-weight (HMW) CS-induced interferon regulatory factor pathway signaling, antigen-presenting cell activation, and cytokine messenger RNA (mRNA) production, with LMW inducing higher mRNA levels at 24 h and HMW elevating mRNA responses at 48 h. LMW and HMW CS also induced adaptive immune responses after vaccination, indicated by enhanced immunoglobulin G production in mice receiving LMW CS and increased CD4 interleukin 4 (IL-4) and IL-2 production in mice receiving HMW CS. Importantly, both LMW and HMW CS adjuvantation reduced morbidity following homologous IAV challenge. Taken together, these results support that LMW and HMW CS can act as adjuvants, although this protection may be mediated through distinct mechanisms based on CS MW.

Combined TLR4 and TLR9 agonists induce distinct phenotypic changes in innate immunity in vitro and in vivo.

Toll-like receptor (TLR)4 and TLR9 agonists, MPL and CpG, are used as adjuvants in vaccines and have been investigated for their combined potential. However, how these two combined agonists regulate transcriptional changes in innate immune cells and cells at the site of vaccination has not been thoroughly investigated. Here, we utilized transcriptomics to investigate how CpG, MPL, and CpG + MPL impact gene expression in dendritic cells (DC) in vitro. Principal component analysis of transcriptional changes after single and combined treatment indicated that CpG, MPL, and CpG + MPL caused distinct gene signatures. CpG + MPL induced antiviral gene expression and activated the interferon regulatory factor pathway. In vitro changes were associated with lower in vivo morbidity upon viral challenge, elevated systemic cytokine protein production, local cytokine mRNA expression, and increased migratory monocyte derived DC populations in the draining lymph node following vaccination with CpG + MPL. This report suggests that CpG + MPL enhances transcription of antiviral and inflammatory genes and increases DC migration.

Analysis of Student Perceptions of Just-In-Time Teaching Pedagogy in PharmD Microbiology and Immunology Courses.

Just-In-Time Teaching (JiTT) active learning pedagogy is utilized by various disciplines, but its value in a professional pharmacy curriculum has not yet been demonstrated. The purpose of our research study is to implement and evaluate JiTT in a Doctor of Pharmacy (PharmD) program. The impetus in implementing JiTT into a PharmD curriculum was to provide students with an out-of-classroom learning opportunity to enhance knowledge-based skills. The current study summarizes the implementation of JiTT in four distinct instances: two iterations of the required courses "Integrated Microbiology and Virology" (Fall 2016 and Fall 2017) and "Integrated Immunology" (Winter 2016-2017 and Winter 2017-2018). JiTT included knowledge-based questions in multiple-choice format, integrated case studies, and student responses prior to the actual lecture session. After the conclusion of each course, students were asked to provide feedback on the utilization of JiTT by way of an anonymous survey. Following the Fall 2016 iteration of the Microbiology & Virology course, students found the integrated case studies to be beneficial (mean = 3.27 out of a maximum of 4, SD = 0.62), and their overall endorsement of JiTT was high (mean = 3.61 out of 4, SD = 0.50). For the other three courses included in this study, the primary dependent variable was the student's average rating of JiTT, rated on a five-point scale. Aggregating the scores from the Fall 2017 iteration of the Integrated Microbiology & Virology course and both instances of the Immunology course, students rated JiTT very favorably (mean = 4.17 out of a maximum of 5, SD = 0.77). Students' performances in JiTT-based courses were compared against non-JiTT-based courses. Analysis of assessment data for student's performance on knowledge-based questions showed JiTT was helpful for student learning and JiTT-based courses had more consistent exam scores compared to non-JiTT-based courses. The current results are a promising initial step in validating the usefulness of JiTT in a pharmacy program and lays the foundation for future studies aimed at a direct comparison between a traditional lecture style and JiTT pedagogy implemented into PharmD curricula.

A Mechanistic Computational Model Reveals That Plasticity of CD4+ T Cell Differentiation Is a Function of Cytokine Composition and Dosage.

CD4+ T cells provide cell-mediated immunity in response to various antigens. During an immune response, naïve CD4+ T cells differentiate into specialized effector T helper (Th1, Th2, and Th17) cells and induced regulatory (iTreg) cells based on a cytokine milieu. In recent studies, complex phenotypes resembling more than one classical T cell lineage have been experimentally observed. Herein, we sought to characterize the capacity of T cell differentiation in response to the complex extracellular environment. We constructed a comprehensive mechanistic (logical) computational model of the signal transduction that regulates T cell differentiation. The model's dynamics were characterized and analyzed under 511 different environmental conditions. Under these conditions, the model predicted the classical as well as the novel complex (mixed) T cell phenotypes that can co-express transcription factors (TFs) related to multiple differentiated T cell lineages. Analyses of the model suggest that the lineage decision is regulated by both compositions and dosage of signals that constitute the extracellular environment. In this regard, we first characterized the specific patterns of extracellular environments that result in novel T cell phenotypes. Next, we predicted the inputs that can regulate the transition between the canonical and complex T cell phenotypes in a dose-dependent manner. Finally, we predicted the optimal levels of inputs that can simultaneously maximize the activity of multiple lineage-specifying TFs and that can drive a phenotype toward one of the co-expressed TFs. In conclusion, our study provides new insights into the plasticity of CD4+ T cell differentiation, and also acts as a tool to design testable hypotheses for the generation of complex T cell phenotypes by various input combinations and dosages.

Oral Non-Viral Gene Delivery for Applications in DNA Vaccination and Gene Therapy.

Non-viral gene delivery via the oral route is a promising strategy for improving outcomes of DNA vaccination and gene therapy applications. Unlike traditional parenteral administration routes, the oral route is a non-invasive approach that lends itself to high patient compliance and ease of dosing. Moreover, oral administration allows for both local and systemic production of therapeutic genes or, in the case of DNA vaccination, mucosal and systemic immunity. However, the oral route presents distinct challenges and barriers to achieving successful gene delivery. Oral non-viral gene delivery systems must be able to survive the harsh and variable environments (e.g. acidic pH, degrading enzymes, mucus layer) encountered during transit through the gastrointestinal tract, while still allowing for efficient transgene production at sites of interest. These barriers present unique design challenges for researchers in material selection and in improving the transfection efficiency of orally delivered genes. This review provides an overview of advancements in the design of oral non-viral gene delivery systems, and highlights recent and important developments towards improving orally delivered genes for applications in gene therapy and DNA vaccination.

Activation of IRF3 contributes to IFN-γ and ISG54 expression during the immune responses to B16F10 tumor growth.

Interferon Regulatory Factor (IRF-3) has been shown to contribute to immune control of B16 melanoma tumor growth. We have shown previously that IRF-3 has a role in IFN-γ-induced expression of pro-apoptotic interferon stimulated gene 54 (ISG54) in macrophages and IFN-γ in T cells. To investigate the IRF3-IFN-γ-ISG54 nexus, we injected C57Bl/6 (B6) and IRF3KO mice s.c. with luciferase-producing B16-F10 tumor cells. Tumor growth as measured by luciferase levels was similar between B6 and IRF3KO mice at days 2 and 6, but was significantly greater at day 9 in IRF3KO mice compared with B6 mice. Transcription factor assays on splenic protein extracts after tumor inoculation revealed peak activation of IRF3 and IRF7 at day 6 in B6 tumor-bearing mice but not in IRF3KO tumor-bearing mice. Likewise, significant induction of IFN-γ occurred in spleens and tumors in B6 mice from days 6-9 but failed to occur in tumor-bearing IRF3KO mice. Previous reports from other labs showed that the anti-tumor properties of IFN-γ are the result of cell cycle arrest. Using B16F1 cells or B16F1 cells deficient in IFN-γ receptor (B16-IRFGRKO), we found that IFN-γ alone and in synergy with the TLR3/IRF3 agonists, poly I:C, decreased B16F1 cell growth in significant correlation with increased ISG54 expression. Moreover, IFN-γ alone increased expression of the cell cycle inhibitor, p27Kip while IFN-γ plus poly I:C increased cleaved Caspase-3 in B16 cells. Thus, it is likely that an IFN-γ/IRF3/ISG54 nexus can significantly contribute to tumor cell control during anti-tumor immune responses.

Chitosan-zein nano-in-microparticles capable of mediating in vivo transgene expression following oral delivery.

The oral route is an attractive delivery route for the administration of DNA-based therapeutics, specifically for applications in gene therapy and DNA vaccination. However, oral DNA delivery is complicated by the harsh and variable conditions encountered throughout gastrointestinal (GI) transit, leading to degradation of the delivery vector and DNA cargo, and subsequent inefficient delivery to target cells. In this work, we demonstrate the development and optimization of a hybrid-dual particulate delivery system consisting of two natural biomaterials, zein (ZN) and chitosan (CS), to mediate oral DNA delivery. Chitosan-Zein Nano-in-Microparticles (CS-ZN-NIMs), consisting of core Chitosan/DNA nanoparticles (CS/DNA NPs) prepared by ionic gelation with sodium tripolyphosphate (TPP), further encapsulated in ZN microparticles, were formulated using a water-in-oil emulsion (W/O). The resulting particles exhibited high CS/DNA NP loading and encapsulation within ZN microparticles. DNA release profiles in simulated gastric fluid (SGF) were improved compared to un-encapsulated CS/DNA NPs. Further, site-specific degradation of the outer ZN matrix and release of transfection competent CS/DNA NPs occurred in simulated intestinal conditions with CS/DNA NP cores successfully mediating transfection in vitro. Finally, CS-ZN-NIMs encoding GFP delivered by oral gavage in vivo induced the production of anti-GFP IgA antibodies, demonstrating in vivo transfection and expression. Together, these results demonstrate the successful formulation of CS-ZN-NIMs and their potential to improve oral gene delivery through improved protection and controlled release of DNA cargo.

Significant role for IRF3 in both T cell and APC effector functions during T cell responses.

Interferon Regulatory Factor (IRF)3 is a crucial transcription factor during innate immune responses. Here we show IRF3 also has a role in adaptive T cell immune responses. Expression of IFN-γ, IL-17, and Granzyme B (GrB) during in vitro T cell responses was impaired when either dendritic cells (DCs) or T cells were derived from IRF3KO mice. Unexpectedly, IRF3-dependent NK-activating molecule (INAM), which is an NK cell activating factor of the DC innate immune response, was induced during the T cell response. Additionally, supernatants from responding T cells induced ISG54 in the RAW264.7 macrophage cell line in an IRF3 dependent manner. Moreover, addition of anti-IFN-γ prevented supernatant induction of ISG54 and recombinant IFN-γ stimulated ISG54 expression. Thus, IRF3 in APCs and T cells is required for optimal T-cell effector function and the ability of T cells to influence innate immune function of APCs.

Micro- and nanoparticulates for DNA vaccine delivery.

DNA vaccination has emerged as a promising alternative to traditional protein-based vaccines for the induction of protective immune responses. DNA vaccines offer several advantages over traditional vaccines, including increased stability, rapid and inexpensive production, and flexibility to produce vaccines for a wide variety of infectious diseases. However, the immunogenicity of DNA vaccines delivered as naked plasmid DNA is often weak due to degradation of the DNA by nucleases and inefficient delivery to immune cells. Therefore, biomaterial-based delivery systems based on micro- and nanoparticles that encapsulate plasmid DNA represent the most promising strategy for DNA vaccine delivery. Microparticulate delivery systems allow for passive targeting to antigen presenting cells through size exclusion and can allow for sustained presentation of DNA to cells through degradation and release of encapsulated vaccines. In contrast, nanoparticle encapsulation leads to increased internalization, overall greater transfection efficiency, and the ability to increase uptake across mucosal surfaces. Moreover, selection of the appropriate biomaterial can lead to increased immune stimulation and activation through triggering innate immune response receptors and target DNA to professional antigen presenting cells. Finally, the selection of materials with the appropriate properties to achieve efficient delivery through administration routes conducive to high patient compliance and capable of generating systemic and local (i.e. mucosal) immunity can lead to more effective humoral and cellular protective immune responses. In this review, we discuss the development of novel biomaterial-based delivery systems to enhance the delivery of DNA vaccines through various routes of administration and their implications for generating immune responses.

The Differentiation and Protective Function of Cytolytic CD4 T Cells in Influenza Infection.

CD4 T cells that recognize peptide antigen in the context of class II MHC can differentiate into various subsets that are characterized by their helper functions. However, increasing evidence indicates that CD4 cells with direct cytolytic activity (CD4 CTL) play a role in chronic as well as acute infections, such as influenza A virus (IAV) infection. In the last couple of decades, techniques to measure the frequency and activity of these cytolytic cells has demonstrated their abundance in infections, such as human immunodeficiency virus, mouse pox, murine gamma herpes virus, cytomegalovirus, Epstein-Barr virus, and influenza among others. We now appreciate a greater role for CD4 CTL as direct effectors in viral infections and antitumor immunity through their ability to acquire perforin-mediated cytolytic activity and contribution to lysis of virally infected targets or tumors. As early as the 1980s, CD4 T cell clones with cytolytic potential were identified after influenza virus infection, yet much of this early work was dependent on in vitro culture and little was known about the physiological relevance of CD4 CTL. Here, we discuss the direct role CD4 CTL play in protection against lethal IAV infection and the factors that drive the generation of perforin-mediated lytic activity in CD4 cells in vivo during IAV infection. While focusing on CD4 CTL generated during IAV infection, we pull comparisons from the literature in other antiviral and antitumor systems. Further, we highlight what is currently known about CD4 CTL secondary and memory responses, as well as vaccination strategies to induce these potent killer cells that provide an extra layer of cell-mediated immune protection against heterosubtypic IAV infection.

Single-Dose CpG Immunization Protects Against a Heterosubtypic Challenge and Generates Antigen-Specific Memory T Cells.

Despite extensive research, influenza A virus (IAV) remains a major cause of morbidity, mortality, and healthcare expenditure. Emerging pandemics from highly pathogenic IAV strains, such as H5N1 and pandemic H1N1, highlight the need for universal, cross-protective vaccines. Current vaccine formulations generate strain-specific neutralizing antibodies primarily against the outer coat proteins, hemagglutinin and neuraminidase. In contrast to these highly mutable proteins, internal proteins of IAV are more conserved and are a favorable target for developing vaccines that induce strong T cell responses in addition to humoral immunity. Here, we found that intranasal administration with a single dose of CpG and inactivated x31 (H3N2) reduced viral titers and partially protected mice from a heterosubtypic challenge with a lethal dose of PR8 (H1N1). Early after immunization, vaccinated mice showed increased innate immune activation with high levels of MHCII and CD86 expression on dendritic cells in both draining lymph nodes and lungs. Three days after immunization, CD4 and CD8 cells in the lung upregulated CD69, suggesting that activated lymphocytes are present at the site of vaccine administration. The ensuing effector Th1 responses were capable of producing multiple cytokines and were present at least 30 days after immunization. Furthermore, functional memory responses were observed, as antigen-specific IFN-γ(+) and GrB(+) cells were detected early after lethal infection. Together, this work provides evidence for using pattern recognition receptor agonists as a mucosal vaccine platform for inducing robust T cell responses capable of protecting against heterologous IAV challenges.

IRF3 deficiency impacts granzyme B expression and maintenance of memory T cell function in response to viral infection.

The role of interferon regulatory factor 3 (IRF3) in the innate immune response to infection has been well studied. However, less is known about IRF3 signaling in shaping the adaptive T cell response. To determine the role of IRF3 in the generation and maintenance of effective anti-viral T cell responses, mice deficient in IRF3 were infected with a potentially persistent virus, Theiler's murine encephalomyelitis virus (TMEV) or with a model acute infection, influenza A virus (IAV). IRF3 was required to prevent TMEV persistence and induce robust TMEV specific effector T cell responses at the site of infection. This defect was more pronounced in the memory phase with an apparent lack of TMEV-specific memory T cells expressing granzyme B (GrB) in IRF3 deficient mice. In contrast, IRF3 had no effect on antigen specific T cell responses at the effector stage during IAV infection. However, memory T cell responses to IAV were also impaired in IRF3 deficient mice. Furthermore, addition of cytokines during peptide restimulation could not restore GrB expression in IRF3 deficient memory T cells. Taken together, IRF3 plays an important role in the maintenance of effective anti-viral T cell memory responses.

Early cytokine dysregulation and viral replication are associated with mortality during lethal influenza infection.

Infection with influenza A virus (IAV) leads to acute lung injury and possibly fatal complications, especially in immunocompromised, elderly, or chronically infected individuals. Therefore, it is important to study the factors that lead to pathology and mortality in infected hosts. In this report, we analyze immune responses to infection at a sublethal (0.1 LD(50)) and lethal (1 LD(50)) dose of the highly pathogenic IAV A/Puerto Rico/8/34 (PR8). Our experiments revealed that infection with a 1 LD(50) dose induced peak viral titers at day 2 compared to day 4 in the 0.1 LD(50) dose. Moreover, early cytokine dysregulation was observed in the lethal dose with significantly elevated levels of IFN-α, TNF-α, CXCL9, IL-6, and MCP-1 produced at day 2. Early inflammatory responses following infection with 1 LD(50) correlated with a greater influx of neutrophils into the lung. However, depletion of neutrophils enhanced morbidity following IAV infection. Though no differences in CD8+ cell function were observed, CD4+ effector responses were impaired in the lungs 8 days after infection with 1 LD(50). Histological analysis revealed significant pathology in lethally infected mice at day 2 and day 6 postinfection, when viral titers remained high. Treating lethally infected mice with oseltamivir inhibited viral titers to sublethal levels, and abrogated the pathology associated with the lethal dose. Together, these results suggest that early cytokine dysregulation and viral replication play a role in pulmonary damage and high mortality in lethally infected mice.

Inflammation enhances IL-2 driven differentiation of cytolytic CD4 T cells.

Cytolytic CD4 T cells (CD4 CTL) have been identified in vivo in response to viral infections; however, the factors necessary for driving the cytolytic phenotype have not been fully elucidated. Our previously published work suggests IL-2 may be the master regulator of perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of IL-2 in CD4 CTL generation, T cell receptor transgenic mice deficient in the ability to produce IL-2 or the high affinity IL-2 receptor (IL-2Rα, CD25) were used. Increasing concentrations of IL-2 were necessary to drive perforin (Prf) expression and maximal cytotoxicity. Granzyme B (GrB) expression and killing correlated with STAT5 activation and CD25 expression in vitro, suggesting that signaling through the high affinity IL-2R is critical for full cytotoxicity. IL-2 signaling was also necessary in vivo for inducing the Th1 phenotype and IFN-γ expression in CD4 T cells during influenza A (IAV) infection. In addition, GrB expression, as measured by mean fluorescent intensity, was decreased in CD25 deficient cells; however, the frequency of CD4 cells expressing GrB was unchanged. Similarly, analysis of cytolytic markers such as CD107a/b and Eomesodermin indicate high IL-2Rα expression is not necessary to drive the CD4 CTL phenotype during IAV infection. Thus, inflammatory signals induced by viral infection may overcome the need for strong IL-2 signals in driving cytotoxicity in CD4 cells.

Interferon response factor 3 is crucial to poly-I:C induced NK cell activity and control of B16 melanoma growth.

Interferon Response Factor 3 (IRF3) induces several NK-cell activating factors, is activated by poly-I:C, an experimental cancer therapeutic, but is suppressed during many viral infections. IRF3 Knockout (KO) mice exhibited enhanced B16 melanoma growth, impaired intratumoral NK cell infiltration, but not an impaired poly-I:C therapeutic effect due to direct suppression of B16 growth. IRF3 was responsible for poly-I:C decrease in TIM-3 expression by intratumoral dendritic cells, induction of NK-cell Granzyme B and IFN-γ, and induction of macrophage IL-12, IL-15, IL-6, and IRF3-dependent NK-activating molecule (INAM). Thus, IRF3 is a key factor controlling melanoma growth through NK-cell activities, especially during poly-I:C therapy.