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Toll-like receptor (TLR) agonists are being developed as anti-cancer therapeutics due to their potent immunostimulatory properties. However, clinical trials testing TLR agonists as monotherapy have often failed to demonstrate significant improvement over standard of care. We hypothesized that the anti-cancer efficacy of TLR agonist immunotherapy could be improved by combinatorial approaches. To prevent increased toxicity, often seen with systemic combination therapies, we developed a hydrogel to deliver TLR agonist combinations at low doses, locally, during cancer debulking surgery. Using tumor models of WEHI 164 and bilateral M3–9-M sarcoma and CT26 colon carcinoma, we assessed the efficacy of pairwise combinations of poly(I:C), R848, and CpG in controlling local and distant tumor growth. We show that combination of the TLR3 agonist poly(I:C) and TLR7/8 agonist R848 drives anti-tumor immunity against local and distant tumors. In addition, combination of local poly(I:C) and R848 sensitized tumors to systemic immune checkpoint blockade, improving tumor control. Mechanistically, we demonstrate that local therapy with poly(I:C) and R848 recruits inflammatory monocytes to the tumor draining lymph nodes early in the anti-tumor response. Finally, we provide proof of concept for intraoperative delivery of poly(I:C) and R848 together via a surgically applicable biodegradable hydrogel. [ABSTRACT FROM AUTHOR]

Melanoma poses a challenge in oncology because of its aggressive nature and limited treatment modalities. The tumour microenvironment (TME) in melanoma contains unique properties such as an immunosuppressive and high-density environment, unusual vasculature, and a high number of stromal and immunosuppressive cells. In recent years, numerous experiments have focused on boosting the immune system to effectively remove malignant cells. Adjuvants, consisting of phytochemicals, toll-like receptor (TLR) agonists, and cytokines, have shown encouraging results in triggering antitumor immunity and augmenting the therapeutic effectiveness of anticancer therapy. These adjuvants can stimulate the maturation of dendritic cells (DCs) and infiltration of cytotoxic CD8+ T lymphocytes (CTLs). Furthermore, nanocarriers can help to deliver immunomodulators and antigens directly to the tumour stroma, thereby improving their efficacy against malignant cells. The remodelling of melanoma TME utilising phytochemicals, agonists, and other adjuvants can be combined with current modalities for improving therapy outcomes. This review article explores the potential of adjuvants, drugs, and their nanoformulations in enhancing the anticancer potency of macrophages, CTLs, and natural killer (NK) cells. Additionally, the capacity of these agents to repress the function of immunosuppressive components of melanoma TME, such as immunosuppressive subsets of macrophages, stromal and myeloid cells will be discussed. [ABSTRACT FROM AUTHOR]

Toll-like receptor (TLR) agonists are being developed as anti-cancer therapeutics due to their potent immunostimulatory properties. However, clinical trials testing TLR agonists as monotherapy have often failed to demonstrate significant improvement over standard of care. We hypothesized that the anti-cancer efficacy of TLR agonist immunotherapy could be improved by combinatorial approaches. To prevent increased toxicity, often seen with systemic combination therapies, we developed a hydrogel to deliver TLR agonist combinations at low doses, locally, during cancer debulking surgery. Using tumor models of WEHI 164 and bilateral M3–9-M sarcoma and CT26 colon carcinoma, we assessed the efficacy of pairwise combinations of poly(I:C), R848, and CpG in controlling local and distant tumor growth. We show that combination of the TLR3 agonist poly(I:C) and TLR7/8 agonist R848 drives anti-tumor immunity against local and distant tumors. In addition, combination of local poly(I:C) and R848 sensitized tumors to systemic immune checkpoint blockade, improving tumor control. Mechanistically, we demonstrate that local therapy with poly(I:C) and R848 recruits inflammatory monocytes to the tumor draining lymph nodes early in the anti-tumor response. Finally, we provide proof of concept for intraoperative delivery of poly(I:C) and R848 together via a surgically applicable biodegradable hydrogel.

Infection with human papillomavirus (HPV) typically leads to cervical cancer, skin related cancers and many other tumors. HPV is mainly responsible for evading immune tumor monitoring in HPV related cancers. Toll like receptors (TLRs) are particular pattern recognition molecules. When the body is facing immune danger, it can lead to innate and direct adaptive immunity. TLR plays an important role in initiating antiviral immune responses. HPV can affect the expression level of TLR and interfere with TLR related signaling pathways, resulting in sustained viral infection and even carcinogenesis. This paper introduces the HPV virus and HPV related cancers. We discussed the present comprehension of TLR, its expression and signaling, as well as its role in HPV infection. We also provided a detailed introduction to immunotherapy methods for HPV related diseases based on TLR agonists. This will provide insights into methods that support the therapeutic method of HPV related conditions with TLR agonists [ABSTRACT FROM AUTHOR]

Although toll-like receptor (TLR) agonists hold great promise as immune modulators for reprogramming the suppressive immune landscape in pancreatic ductal adenocarcinoma (PDAC), their use is limited by poor pharmacokinetics (PK) and off-target systemic inflammatory effects. To overcome these challenges as well as to attain drug synergy, we developed a lipid bilayer (LB)-coated mesoporous silica nanoparticle (silicasome) platform for co-delivery of the TLR7/8 agonist 3M-052 with the immunogenic chemotherapeutic agent irinotecan. This was accomplished by incorporating the C18 lipid tail of 3M-052 in the coated LB, also useful for irinotecan remote loading in the porous interior. Not only did the co-formulated carrier improve PK, but it strengthened the irinotecan-induced immunogenic cell death response by 3M-052-mediated dendritic cell activation at the tumor site as well as participating lymph nodes. The accompanying increase in CD8+ T-cell infiltration along with a reduced number of regulatory T-cells was associated with tumor shrinkage and metastasis disappearance in subcutaneous and orthotopic KRAS-mediated pancreatic carcinoma tumor models. Moreover, this therapeutic outcome was accomplished without drug or nanocarrier toxicity. All considered, dual-delivery strategies that combine chemo-immunotherapy with co-formulated TLR agonists or other lipid-soluble immune modulators predict successful intervention in heterogeneous PDAC immune landscapes.

Abstract Background Autologous tumor cell-based vaccines (ATVs) aim to prevent and treat tumor metastasis by activating patient-specific tumor antigens to induce immune memory. However, their clinical efficacy is limited. Mannan-BAM (MB), a pathogen-associated molecular pattern (PAMP), can coordinate an innate immune response that recognizes and eliminates mannan-BAM-labeled tumor cells. TLR agonists and anti-CD40 antibodies (TA) can enhance the immune response by activating antigen-presenting cells (APCs) to present tumor antigens to the adaptive immune system. In this study, we investigated the efficacy and mechanism of action of rWTC-MBTA, an autologous whole tumor cell vaccine consisting of irradiated tumor cells (rWTC) pulsed with mannan-BAM, TLR agonists, and anti-CD40 antibody (MBTA), in preventing tumor metastasis in multiple animal models. Methods The efficacy of the rWTC-MBTA vaccine was evaluated in mice using breast (4T1) and melanoma (B16-F10) tumor models via subcutaneous and intravenous injection of tumor cells to induce metastasis. The vaccine’s effect was also assessed in a postoperative breast tumor model (4T1) and tested in autologous and allogeneic syngeneic breast tumor models (4T1 and EMT6). Mechanistic investigations included immunohistochemistry, immunophenotyping analysis, ELISA, tumor-specific cytotoxicity testing, and T-cell depletion experiments. Biochemistry testing and histopathology of major tissues in vaccinated mice were also evaluated for potential systemic toxicity of the vaccine. Results The rWTC-MBTA vaccine effectively prevented metastasis and inhibited tumor growth in breast tumor and melanoma metastatic animal models. It also prevented tumor metastasis and prolonged survival in the postoperative breast tumor animal model. Cross-vaccination experiments revealed that the rWTC-MBTA vaccine prevented autologous tumor growth, but not allogeneic tumor growth. Mechanistic data demonstrated that the vaccine increased the percentage of antigen-presenting cells, induced effector and central memory cells, and enhanced CD4+ and CD8+ T-cell responses. T-cells obtained from mice that were vaccinated displayed tumor-specific cytotoxicity, as shown by enhanced tumor cell killing in co-culture experiments, accompanied by increased levels of Granzyme B, TNF-α, IFN-γ, and CD107a in T-cells. T-cell depletion experiments showed that the vaccine’s antitumor efficacy depended on T-cells, especially CD4+ T-cells. Biochemistry testing and histopathology of major tissues in vaccinated mice revealed negligible systemic toxicity of the vaccine. Conclusion The rWTC-MBTA vaccine demonstrated efficacy in multiple animal models through T-cell mediated cytotoxicity and has potential as a therapeutic option for preventing and treating tumor metastasis with minimal systemic toxicity.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed in various immune cell types and perform multiple purposes and duties involved in the induction of innate and adaptive immunity. Their capability to propagate immunity makes them attractive targets for the expansion of numerous immunotherapeutic approaches targeting cancer. These immunotherapeutic strategies include using TLR ligands/agonists as monotherapy or combined therapeutic strategies. Several TLR agonists have demonstrated significant efficacy in advanced clinical trials. In recent years, multiple reports established the applicability of TLR agonists as adjuvants to chemotherapeutic drugs, radiation, and immunotherapies, including cancer vaccines. Cancer vaccines are a relatively novel approach in the field of cancer immunotherapy and are currently under extensive evaluation for treating different cancers. In the present review, we tried to deliver an inclusive discussion of the significant TLR agonists and discussed their application and challenges to their incorporation into cancer immunotherapy approaches, particularly highlighting the usage of TLR agonists as functional adjuvants to cancer vaccines. Finally, we present the translational potential of rWTC-MBTA vaccination [irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists Mannan-BAM, TLR ligands, and anti-CD40 agonisticAntibody], an autologous cancer vaccine leveraging membranebound Mannan-BAM, and the immune-inducing prowess of TLR agonists as a probable immunotherapy in multiple cancer types. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed in various immune cell types and perform multiple purposes and duties involved in the induction of innate and adaptive immunity. Their capability to propagate immunity makes them attractive targets for the expansion of numerous immunotherapeutic approaches targeting cancer. These immunotherapeutic strategies include using TLR ligands/agonists as monotherapy or combined therapeutic strategies. Several TLR agonists have demonstrated significant efficacy in advanced clinical trials. In recent years, multiple reports established the applicability of TLR agonists as adjuvants to chemotherapeutic drugs, radiation, and immunotherapies, including cancer vaccines. Cancer vaccines are a relatively novel approach in the field of cancer immunotherapy and are currently under extensive evaluation for treating different cancers. In the present review, we tried to deliver an inclusive discussion of the significant TLR agonists and discussed their application and challenges to their incorporation into cancer immunotherapy approaches, particularly highlighting the usage of TLR agonists as functional adjuvants to cancer vaccines. Finally, we present the translational potential of rWTC-MBTA vaccination [irradiated whole tumor cells (rWTC) pulsed with phagocytic agonists Mannan-BAM, TLR ligands, and anti-CD40 agonisticAntibody], an autologous cancer vaccine leveraging membrane-bound Mannan-BAM, and the immune-inducing prowess of TLR agonists as a probable immunotherapy in multiple cancer types.

Innate immune memory allows macrophages to adequately respond to pathogens to which they have been pre-exposed. To what extent different pattern recognition receptors, cytokines and resolution signals influence innate immune memory needs further elucidation. The present study assessed whether lipopolysaccharide (LPS) tolerance in monocytes and macrophages is affected by these factors. Human CD14+ cells were isolated from peripheral blood, stimulated by LPS and re-stimulated after 3 days of resting. Hereafter, immune-responsive gene 1 (IRG-1), heme oxygenase 1 (HO-1), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expression were assessed. Our study revealed the following findings: (1) While pre-stimulation with the Toll-like receptor 4 ligand LPS inhibits the induction of IRG-1, TNF-α and IL-6 expression, pre-stimulation with TLR 1/2 ligands only affects cytokine production but not IRG-1 expression upon subsequent TLR4 engagement. (2) Prior TNF-α stimulation does not affect LPS tolerance but rather increases LPS-mediated cytokine expression. (3) Dimethyl itaconate (DMI) inhibits the expression of IRG-1 in a dose-dependent manner but does not affect TNF-α or IL-6 expression. (4) Docosahexaenoic acid (DHA) partly inhibits IRG-1 expression in monocytes but not in M(IFNγ) and M(IL-4) polarized macrophages. LPS tolerance is not affected in these cells by DHA. The data presented in this study partly corroborate and extend previous findings on innate immune memory and warrant further studies on LPS tolerance to gain a better understanding of innate immune memory at the molecular level. [ABSTRACT FROM AUTHOR]

Background: Autologous tumor cell-based vaccines (ATVs) aim to prevent and treat tumor metastasis by activating patient-specific tumor antigens to induce immune memory. However, their clinical efficacy is limited. Mannan-BAM (MB), a pathogen-associated molecular pattern (PAMP), can coordinate an innate immune response that recognizes and eliminates mannan-BAM-labeled tumor cells. TLR agonists and anti-CD40 antibodies (TA) can enhance the immune response by activating antigen-presenting cells (APCs) to present tumor antigens to the adaptive immune system. In this study, we investigated the efficacy and mechanism of action of rWTC-MBTA, an autologous whole tumor cell vaccine consisting of irradiated tumor cells (rWTC) pulsed with mannan-BAM, TLR agonists, and anti-CD40 antibody (MBTA), in preventing tumor metastasis in multiple animal models. Methods: The efficacy of the rWTC-MBTA vaccine was evaluated in mice using breast (4T1) and melanoma (B16-F10) tumor models via subcutaneous and intravenous injection of tumor cells to induce metastasis. The vaccine's effect was also assessed in a postoperative breast tumor model (4T1) and tested in autologous and allogeneic syngeneic breast tumor models (4T1 and EMT6). Mechanistic investigations included immunohistochemistry, immunophenotyping analysis, ELISA, tumor-specific cytotoxicity testing, and T-cell depletion experiments. Biochemistry testing and histopathology of major tissues in vaccinated mice were also evaluated for potential systemic toxicity of the vaccine. Results: The rWTC-MBTA vaccine effectively prevented metastasis and inhibited tumor growth in breast tumor and melanoma metastatic animal models. It also prevented tumor metastasis and prolonged survival in the postoperative breast tumor animal model. Cross-vaccination experiments revealed that the rWTC-MBTA vaccine prevented autologous tumor growth, but not allogeneic tumor growth. Mechanistic data demonstrated that the vaccine increased the percentage of antigen-presenting cells, induced effector and central memory cells, and enhanced CD4+ and CD8+ T-cell responses. T-cells obtained from mice that were vaccinated displayed tumor-specific cytotoxicity, as shown by enhanced tumor cell killing in co-culture experiments, accompanied by increased levels of Granzyme B, TNF-α, IFN-γ, and CD107a in T-cells. T-cell depletion experiments showed that the vaccine's antitumor efficacy depended on T-cells, especially CD4+ T-cells. Biochemistry testing and histopathology of major tissues in vaccinated mice revealed negligible systemic toxicity of the vaccine. Conclusion: The rWTC-MBTA vaccine demonstrated efficacy in multiple animal models through T-cell mediated cytotoxicity and has potential as a therapeutic option for preventing and treating tumor metastasis with minimal systemic toxicity. [ABSTRACT FROM AUTHOR]

Summary: Background: Plasmacytoid dendritic cells (pDCs) sense viral and bacterial products through Toll-like receptor (TLR)-7 and -9 and translate this sensing into Interferon-α (IFN-α) production and T-cell activation. The understanding of the mechanisms involved in pDCs stimulation may contribute to HIV-cure immunotherapeutic strategies. The objective of the present study was to characterize the immunomodulatory effects of TLR agonist stimulations in several HIV-1 disease progression phenotypes and in non HIV-1 infected donors. Methods: pDCs, CD4 and CD8 T-cells were isolated from 450 ml of whole blood from non HIV-1 infected donors, immune responders (IR), immune non responders (INR), viremic (VIR) and elite controller (EC) participants. pDCs were stimulated overnight with AT-2, CpG-A, CpG-C and GS-9620 or no stimuli. After that, pDCs were co-cultured with autologous CD4 or CD8 T-cells and with/without HIV-1 (Gag peptide pool) or SEB (Staphylococcal Enterotoxin B). Cytokine array, gene expression and deep immunophenotyping were assayed. Findings: pDCs showed an increase of activation markers levels, interferon related genes, HIV-1 restriction factors and cytokines levels after TLR stimulation in the different HIV-disease progression phenotypes. This pDC activation was prominent with CpG-C and GS-9620 and induced an increase of HIV-specific T-cell response even in VIR and INR comparable with EC. This HIV-1 specific T-cell response was associated with the upregulation of HIV-1 restriction factors and IFN-α production by pDC. Interpretation: These results shed light on the mechanisms associated with TLR-specific pDCs stimulation associated with the induction of a T-cell mediated antiviral response which is essential for HIV-1 eradication strategies. Funding: This work was supported by Gilead fellowship program, the Instituto de Salud Carlos III (Fondo Europeo de Desarrollo Regional, FEDER, “a way to make Europe”) and the Red Temática de Investigación Cooperativa en SIDA and by the Spanish National Research Council (CSIC).

Simple Summary: Polymorphonuclear neutrophils belong to the first line of defense with major contributions to the early innate recognition and elimination of pathogens. The development of effective vaccines for camel infectious diseases will profit from understanding the interaction mechanisms between innate immune cells and pathogen-associated molecular patterns (PAMPs), such as toll-like receptor (TLR) ligands. The present study investigated the impact of the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC on the phenotype and function of camel blood neutrophils. The analysis of stimulation-induced changes in neutrophil cell size, their phagocytosis and ROS production activity, the expression of cell surface antigens, and cell viability identified agonist-specific modulatory effects on camel blood neutrophils. Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCIIhigh fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs), as a family of pattern recognition receptors, play an important role in the recognition of HIV-1 molecular structures by various cells of the innate immune system, but also provide a functional association with subsequent mechanisms of adaptive immunity. TLR7 and TLR8 play a particularly important role in the innate immune response to RNA viruses due to their ability to recognise GU-rich single-stranded RNA molecules and subsequently activate intracellular signalling pathways resulting in expression of genes coding for various biological response modifiers (interferons, proinflammatory cytokines, chemokines). The aim of this review is to summarise the most recent knowledge on the role of TLRs in the innate immune response to HIV-1 and the role of TLR gene polymorphisms in the biology and in the clinical aspects of HIV infections. In addition, the role of TLR agonists as latency reversing agents in research to treat HIV infections and as immunomodulators in HIV vaccine research will be discussed. [ABSTRACT FROM AUTHOR]

Immunogenic cell death (ICD) plays an important role in sensitizing tumor cells to antigen-presenting cells followed by antitumor immunity. However, a successful antitumor response by ICD requires both apoptotic tumor microenvironments and activated immune systems. Ivermectin (IVM) has been shown to induce cell apoptosis through autophagy which can be a great candidate for ICD therapy. However, a single treatment of IVM-free drug is not an ideal anticancer therapy due to its anti-inflammatory effects and cytotoxicity. In the present study, IVM was shown to enhance the ICD process in addition to the treatment of resiquimod (R848), a TLR7/8 agonist, when co-loaded in a squalene-based nanoemulsion (NE). R848-IVM co-loaded NE was developed and characterized in vitro. Antitumor activity of R848-IVM NE was also evaluated in vitro and in vivo. R848-IVM NE exhibited long-term stability and reduced cytotoxicity by IVM. In vivo studies demonstrated that IVM significantly augments the ICD by upregulating Cd8a and releasing HMGB1 in tumor tissue, which could enhance R848-driven antitumor immunity. R848-IVM NE treatment showed strong antitumor activity with over 80% tumor growth inhibition, suggesting a potential combination therapy of systemic co-delivering IVM with TLR agonists against solid cancer. [Display omitted] [ABSTRACT FROM AUTHOR]

Lymph nodes are crucial immune foci as the primary target for cancer immunotherapy. However, the anti-tumor functions of tumor-draining lymph nodes (TDLNs) are critically suppressed by tumors. Here, a novel spatiotemporal nano-regulator is presented, designed to modulate the dendritic cells (DCs) in TDLNs, establishing a supportive niche for immune surveillance. The DC-mediated nano-regulator (DNR) is established by the self-assembly of an imidazoquinoline (IMDQ) prodrug, inhibitory immune checkpoint (ICP) siRNA, and mannan (a TLR4 agonist). This unique design leverages the spatiotemporal activation of TLR4 and TLR7/8, thereby optimizing DC maturation and cytokine production. This further promotes efficient T cell priming. Simultaneously, the ICP-targeting siRNA mitigates the tolerogenic effects induced by tumor-derived factors and TLR activation, preventing T cell exhaustion. In essence, DNR facilitates potent remodeling of TDLNs and the tumor microenvironment, activating the anti-tumor immunity cascade. When combined with vaccines, DNR greatly promotes tumor regression and the establishment of long-term immunological memory., (© 2024 Wiley‐VCH GmbH.)

Toll-like receptors (TLRs) are natural initial triggers of innate and adaptive immune responses. With the advent of cancer immunotherapy, nucleic acids engineered as ligands of endosomal TLRs have been investigated for the treatment of solid tumors. Despite promising results, their systemic administration, similarly to other immunotherapies, raises safety issues. To overcome these problems, recent studies have applied the direct injection of endosomal TLR agonists in the tumor and/or draining lymph nodes, achieving high local drug exposure and strong antitumor response. Importantly, intratumoral delivery of TLR agonists showed powerful effects not only against the injected tumors but also often against uninjected lesions (abscopal effects), resulting in some cases in cure and antitumoral immunological memory. Herein, we describe the structure and function of TLRs and their role in the tumor microenvironment. Then, we provide our vision on the potential of intratumor versus systemic delivery or vaccination approaches using TLR agonists, also considering the use of nanoparticles to improve their targeting properties. Finally, we collect the preclinical and clinical studies applying intratumoral injection of TLR agonists as monotherapies or in combination with: (a) other TLR or STING agonists; (b) other immunotherapies; (c) radiotherapy or chemotherapy; (d) targeted therapies. [ABSTRACT FROM AUTHOR]

• Nicotine compromises humoral response to a protein antigen mixed with Alum. • Nicotine compromises cellular response to a protein antigen mixed with TLR4 agonist. • TLR3 or TLR7/8 agonist diminishes the adverse effects of nicotine on immunization. • The increased adjuvanticity of TLR3 agonist is not dependent on DC-NK cross-talk. • The increased adjuvanticity of TLR7/8 agonist is dependent on DC-NK cross-talk. It is known that cigarette smoke compromises the immune system and increases the risk of vaccine-preventable diseases. We reported that nicotine, the immunosuppressive component of cigarette smoke, disrupts the functional properties of DC and DC crosstalk with NK cells, which is pivotal in the initiation of immune responses to vaccines. We also showed that select TLR agonists could reduce the degrading effects of nicotine on DC-NK mediated immune responses in vitro. In this study, we further investigated the magnitude and mechanism of immune responses to a protein antigen formulated with alum or TLR agonists using WT, NK-depleted, and IFN-γ deficient mice exposed to nicotine. We found that R848 followed by Poly(I:C) acted as the most effective adjuvants to increase the percentage and number of IFN-γ-producing effector NK cells in the lymph nodes of immunized mice. In addition, we observed that the protein antigen formulated with Poly(I:C) or R848 improved the antigen-specific immune response in nicotine-exposed mice through NK-independent and -dependent mechanisms, respectively. These findings extend our understanding of the hyporesponsiveness of smokers to vaccines and provides the means to increase the efficacy of vaccines in this large population. [Display omitted] [ABSTRACT FROM AUTHOR]

Cancer vaccines offer the potential to enhance T cell-mediated antitumor immunity by expanding and increasing the function of tumor-specific T cells and shaping the recall response against recurring tumors. While the use of cancer vaccines is not a new immunotherapeutic approach, the cancer vaccine field continues to evolve as new antigen types emerge and vaccine formulations and delivery strategies are developed. As monotherapies, cancer vaccines have not been very efficacious in part due to pre-existing peripheral- and tumor-mediated tolerance mechanisms that limit T cell function. Over the years, various agents including Toll-like receptor agonists, cytokines, and checkpoint inhibitors have been employed as vaccine adjuvants and immune modulators to increase antigen-mediated activation, expansion, memory formation, and T effector cell function. A renewed interest in this approach has emerged as better neoepitope discovery tools are being developed and our understanding of what constitutes an effective cancer vaccine is improved. In the coming years, cancer vaccines will likely be vital to enhance the response to current immunotherapies. In this review, we discuss the various types of therapeutic cancer vaccines, including types of antigens and approaches used to enhance cancer vaccine responses such as TLR agonists, recombinant interleukin-2 and interleukin-2 derivatives, and checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

NK cells are well‐known for their antiviral functions. Also, their role in HIV has been well established, with rapid responses elicited during early HIV infection. Most immune cells including CD4+ T cells, monocytes, Mϕs, and dendritic cells are readily infected by HIV. Recent evidence from multiple studies has suggested that similar to these cells, in chronic conditions like HIV, NK cells also undergo functional exhaustion with impaired cytotoxicity, altered cytokine production, and impaired ADCC. NK‐based immunotherapy aims to successfully restore, boost, and modify their activity as has been already demonstrated in the field of cancer immunotherapy. The utilization of NK cell‐based strategies for the eradication of HIV from the body provides many advantages over classical ART. The literature search consisted of manually selecting the most relevant studies from databases including PubMed, Embase, Google Scholar, and ClinicalTrial.gov. Some of the treatments currently under consideration are CAR‐NK cell therapy, facilitating ADCC, TLR agonists, bNAbs, and BiKEs/TriKEs, blocking inhibitory NK receptors during infection, IL‐15 and IL‐15 superagonists (eg: ALT‐803), and so on. This review aims to discuss the NK cell‐based therapies currently under experimentation against HIV infection and finally highlight the challenges associated with NK cell‐based immunotherapies. [ABSTRACT FROM AUTHOR]

Cancer vaccines offer the potential to enhance T cell-mediated antitumor immunity by expanding and increasing the function of tumor-specific T cells and shaping the recall response against recurring tumors. While the use of cancer vaccines is not a new immunotherapeutic approach, the cancer vaccine field continues to evolve as new antigen types emerge and vaccine formulations and delivery strategies are developed. As monotherapies, cancer vaccines have not been very efficacious in part due to pre-existing peripheral- and tumor-mediated tolerance mechanisms that limit T cell function. Over the years, various agents including Toll-like receptor agonists, cytokines, and checkpoint inhibitors have been employed as vaccine adjuvants and immune modulators to increase antigen-mediated activation, expansion, memory formation, and T effector cell function. A renewed interest in this approach has emerged as better neoepitope discovery tools are being developed and our understanding of what constitutes an effective cancer vaccine is improved. In the coming years, cancer vaccines will likely be vital to enhance the response to current immunotherapies. In this review, we discuss the various types of therapeutic cancer vaccines, including types of antigens and approaches used to enhance cancer vaccine responses such as TLR agonists, recombinant interleukin-2 and interleukin-2 derivatives, and checkpoint inhibitors.

Combined antiretroviral therapy (cART) can inhibit the replication of human immunodeficiency virus type 1 (HIV-1) and reduce viral loads in the peripheral blood to undetectable levels. However, the presence of latent HIV-1 reservoirs prevents complete HIV-1 eradication. Several drugs and strategies targeting T cells are now in clinical trials, but their effectiveness in reducing viral reservoirs has been mixed. Interestingly, innate immune natural killer (NK) cells, which are promising targets for cancer therapy, also play an important role in HIV-1 infection. NK cells are a unique innate cell population with features of adaptive immunity that can regulate adaptive and innate immune cell populations; therefore, they can be exploited for HIV-1 immunotherapy and reservoir eradication. In this review, we highlight immunotherapy strategies for HIV infection that utilize the beneficial properties of NK cells. [ABSTRACT FROM AUTHOR]

Combined antiretroviral therapy (cART) can inhibit the replication of human immunodeficiency virus type 1 (HIV-1) and reduce viral loads in the peripheral blood to undetectable levels. However, the presence of latent HIV-1 reservoirs prevents complete HIV-1 eradication. Several drugs and strategies targeting T cells are now in clinical trials, but their effectiveness in reducing viral reservoirs has been mixed. Interestingly, innate immune natural killer (NK) cells, which are promising targets for cancer therapy, also play an important role in HIV-1 infection. NK cells are a unique innate cell population with features of adaptive immunity that can regulate adaptive and innate immune cell populations; therefore, they can be exploited for HIV-1 immunotherapy and reservoir eradication. In this review, we highlight immunotherapy strategies for HIV infection that utilize the beneficial properties of NK cells.

Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCIIhigh fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality.

Toll-like receptors (TLRs), as a family of pattern recognition receptors, play an important role in the recognition of HIV-1 molecular structures by various cells of the innate immune system, but also provide a functional association with subsequent mechanisms of adaptive immunity. TLR7 and TLR8 play a particularly important role in the innate immune response to RNA viruses due to their ability to recognise GU-rich single-stranded RNA molecules and subsequently activate intracellular signalling pathways resulting in expression of genes coding for various biological response modifiers (interferons, proinflammatory cytokines, chemokines). The aim of this review is to summarise the most recent knowledge on the role of TLRs in the innate immune response to HIV-1 and the role of TLR gene polymorphisms in the biology and in the clinical aspects of HIV infections. In addition, the role of TLR agonists as latency reversing agents in research to treat HIV infections and as immunomodulators in HIV vaccine research will be discussed.

Background: Glioma, characterized by limited lymphocytic infiltration, constitutes an "immune-desert" tumor displaying insensitivity to various immunotherapies. This study aims to explore therapeutic strategies for inducing tertiary lymphoid structure (TLS) formation within the glioma microenvironment (GME) to transition it from an immune-resistant to an activated state., Methods: TLS formation in GME was successfully induced by intracranial administration of Toll-like receptor (TLR) agonists (OK-432, TLR2/4/9 agonist) and glioma antigens (i.c. αTLR-mix). We employed staining analysis, antibody neutralization, single-cell RNA sequencing (scRNA-Seq), and BCR/TCR sequencing to investigate the underlying mechanisms of TLS formation and its role in anti-glioma immunity. Additionally, a preliminary translational clinical study was conducted., Results: TLS formation correlated with increased lymphocyte infiltration in GME and led to improved prognosis in glioma-bearing mice. In the study of TLS induction mechanisms, certain macrophages/microglia and Th17 displayed markers of "LTo" and "LTi" cells, respectively, interaction through LTα/β-LTβR promoted TLS induction. Post-TLS formation, CD4+ and CD8+ T cells but not CD19+ B cells contributed to anti-glioma immunity. Comparative analysis of B/T cells between brain and lymph node showed that brain B/T cells unveiled switch from naïve to mature, some B cells highlighted an enrichment of CSR-associated genes, V gene usage and clonotype bias were observed. In related clinical studies, i.c. αTLR-mix treatment exhibited tolerability, and chemokines/cytokines assay provided preliminary evidence supporting TLS formation in GME., Conclusion: TLS induction in GME enhanced anti-glioma immunity, improved the immune microenvironment, and controlled glioma growth, suggesting potential therapeutic avenues for treating glioma in the future., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Introduction: Cervical cancer is one of main causes of cancer death in women, especially in developing countries. Therefore, a low-cost broad-spectrum preventive vaccine is immediately needed. The RG-1 epitope of L2 protein is a major crossneutralizing epitope but has low immunogenicity. This defect can be overcome by using built-in adjuvants such as TLR agonists and bacterial toxoid epitopes. To address this issue, we designed an epitope-based vaccine against HPV16 using immunoinformatic tools. Methods: The HPV16 RG-1 epitope was linked to built-in adjuvants including the D1 domain of flagellin and RS09 epitope as TLR agonists, and a tetanus toxoid epitope for induction of immune responses. Using immunoinformatic tools, the immunological characteristics of the construct were evaluated. In the first step, MHC-I and II binding, CD4+ T cell immunogenicity prediction, and in the second step, immunogenicity simulation of the construct were investigated. Results: MHC-I and II predicted epitopes showed a high potentiality to bind to mice and human MHC alleles. The results of the binding of the RG-1 epitope to MHC-I and MHC-II showed that RG-1 could induce humoral and cellular immune response while fused to three built-in adjuvants. Also, the CD4+ immunogenicity assessment results predicted that several epitopes in the designed construct, including epitopes of D1 domain and tetanus toxoid P2 epitope, behaved as potentially strong Th inducers. The immunogenicity simulation results showed that the construct could potentially provide sufficient antigen, and induce suitable humoral and cellular immune responses. Conclusion: The development of new vaccine strategies has been the focus of several studies. The results showed that the designed construct can potentially provide an effective model for developing a preventive vaccine candidate against a variety of HPV genotypes. [ABSTRACT FROM AUTHOR]

A comprehensive SAR study of a putative TLR 3/8/9 agonist was conducted. Despite the excitement surrounding the potential of the first small molecule TLR3 agonist with a compound that additionally displayed agonist activity for TLR8 and TLR9, compound 1 displayed disappointing activity in our hands, failing to match the potency (EC50) reported and displaying only a low efficacy for the extent of stimulated NF-κB activation and release. The evaluation of >75 analogs of 1, many of which constitute minor modifications in the structure, failed to identify any that displayed significant activity and none that exceeded the modest activity found for 1. [ABSTRACT FROM AUTHOR]

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion ("IVAX-1") was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD. • Adjuvanted ebolavirus glycoproteins elicit cross-reactive antibodies and T cells. • Breadth of antibody and T cell responses is increased by multiplexing glycoproteins. • T cell responses were enhanced by delivery on lipid nanoparticles. [ABSTRACT FROM AUTHOR]

Current methodologies for assessing vaccine effectiveness and longevity primarily center on measuring vaccine-induced neutralizing antibodies in serum or plasma. However, these methods overlook additional parameters such as the presence of memory B cells, even as antibody levels wane, and the pivotal role played by memory T cells in shaping antigen-specific memory B cell responses. Several studies have employed a combination of polyclonal activators, such as CpG and R848, along with various cytokines to provoke the recall of memory B cells from peripheral blood mononuclear cells (PBMCs) into antibody-secreting cells (ASCs). Other studies have examined the use of live attenuated viruses to stimulate antigen-specific memory T cells within PBMCs into effector T cells that produce Th1/Th2 cytokines. However, these studies have not fully elucidated the distinct effects of these polyclonal activators on individual subsets, nor have they evaluated whether the vaccine antigen alone is sufficient to trigger the recall of memory T cells. Thus, in this study, we directly compared the capacity of two B cell polyclonal activators to induce the transition of existing vaccine-specific memory cells present in peripheral blood samples into ASCs. Simultaneously, we also assessed the transition of existing memory T cells into effector subsets in response to vaccine antigens. Our findings demonstrate that both polyclonal activator combinations, CpG with IL-6 and IL-15, as well as R848 with IL-2, effectively induce the terminal differentiation of memory B cells into ASCs. Notably, CpG treatment preferentially expanded naïve and non-class-switched B cells, while R848 expanded class-switched memory cells, plasmablasts, and plasma cells. Consequently, R848 treatment led to a greater overall production of total and antigen-specific IgG immunoglobulins. Additionally, the exposure of isolated PBMCs to vaccine antigens alone proved sufficient for recalling the rare antigen-specific memory T cells into effector subsets, predominantly consisting of IFN-γ-producing CD4 T cells and TNF-β-producing CD8 T cells. This study not only establishes a rationale for the selection of methods to expand and detect antigen-specific lymphocyte subsets but also presents a means to quantify vaccine effectiveness by correlating serum antibody levels with preexisting memory cells within peripheral blood samples. [Display omitted] • CpG + IL-6 + IL-15 preferentially expands total, naïve, and nonclass-switched B cells. • R848 + IL-2 optimally expands class-switched, plasmablasts, and plasma cells. • R848 + IL-2 also enhances the production of total and antigen-specific IgG. • Exposure of PBMC to vaccine antigen alone recalls childhood memory T cells. • IFN-γ + CD4+ and TNF-β + CD8+ T cells are majority of recalled childhood memory T cells. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are natural initial triggers of innate and adaptive immune responses. With the advent of cancer immunotherapy, nucleic acids engineered as ligands of endosomal TLRs have been investigated for the treatment of solid tumors. Despite promising results, their systemic administration, similarly to other immunotherapies, raises safety issues. To overcome these problems, recent studies have applied the direct injection of endosomal TLR agonists in the tumor and/or draining lymph nodes, achieving high local drug exposure and strong antitumor response. Importantly, intratumoral delivery of TLR agonists showed powerful effects not only against the injected tumors but also often against uninjected lesions (abscopal effects), resulting in some cases in cure and antitumoral immunological memory. Herein, we describe the structure and function of TLRs and their role in the tumor microenvironment. Then, we provide our vision on the potential of intratumor versus systemic delivery or vaccination approaches using TLR agonists, also considering the use of nanoparticles to improve their targeting properties. Finally, we collect the preclinical and clinical studies applying intratumoral injection of TLR agonists as monotherapies or in combination with: (a) other TLR or STING agonists; (b) other immunotherapies; (c) radiotherapy or chemotherapy; (d) targeted therapies.

Despite advances in critical care medicine, infection remains a significant problem that continues to be complicated with the challenge of antibiotic resistance. Immunocompromised patients are highly susceptible to development of severe infection which often progresses to the life-threatening condition of sepsis. Thus, immunotherapies aimed at boosting host immune defenses are highly attractive strategies to ward off infection and protect patients. Recently there has been mounting evidence that activation of the innate immune system can confer long-term functional reprogramming whereby innate leukocytes mount more robust responses upon secondary exposure to a pathogen for more efficient clearance and host protection, termed trained immunity. Toll-like receptor (TLR) agonists are a class of agents which have been shown to trigger the phenomenon of trained immunity through metabolic reprogramming and epigenetic modifications which drive profound augmentation of antimicrobial functions. Immunomodulatory TLR agonists are also highly beneficial as vaccine adjuvants. This review provides an overview on TLR signaling and our current understanding of TLR agonists which show promise as immunotherapeutic agents for combating infection. A brief discussion on our current understanding of underlying mechanisms is also provided. Although an evolving field, TLR agonists hold strong therapeutic potential as immunomodulators and merit further investigation for clinical translation.

Despite advances in critical care medicine, infection remains a significant problem that continues to be complicated with the challenge of antibiotic resistance. Immunocompromised patients are highly susceptible to development of severe infection which often progresses to the life-threatening condition of sepsis. Thus, immunotherapies aimed at boosting host immune defenses are highly attractive strategies to ward off infection and protect patients. Recently there has been mounting evidence that activation of the innate immune system can confer long-term functional reprogramming whereby innate leukocytes mount more robust responses upon secondary exposure to a pathogen for more efficient clearance and host protection, termed trained immunity. Toll-like receptor (TLR) agonists are a class of agents which have been shown to trigger the phenomenon of trained immunity through metabolic reprogramming and epigenetic modifications which drive profound augmentation of antimicrobial functions. Immunomodulatory TLR agonists are also highly beneficial as vaccine adjuvants. This review provides an overview on TLR signaling and our current understanding of TLR agonists which show promise as immunotherapeutic agents for combating infection. A brief discussion on our current understanding of underlying mechanisms is also provided. Although an evolving field, TLR agonists hold strong therapeutic potential as immunomodulators and merit further investigation for clinical translation. [ABSTRACT FROM AUTHOR]

The development of successful vaccines has been increasingly reliant on the use of immunoadjuvants - additives, which can enhance and modulate immune responses to vaccine antigens. Immunoadjuvants of the polyphosphazene family encompass synthetic biodegradable macromolecules, which attain in vivo activity via antigen delivery and immunostimulation mechanisms. Over the last decades, the technology has witnessed evolvement of next generation members, expansion to include various antigens and routes of administration, and progression to clinical phase. This was accompanied by gaining important insights into the mechanism of action and the development of a novel class of virus-mimicking nano-assemblies for antigen delivery. The present review evaluates in vitro and in vivo data generated to date in the context of latest advances in understanding the primary function and biophysical behavior of these macromolecules. It also provides an overview of relevant synthetic and characterization methods, macromolecular biodegradation pathways, and polyphosphazene-based multi-component, nanoparticulate, and microfabricated formulations. Unlabelled Image • Polyphosphazenes are potent immunostimulants and vaccine delivery vehicles. • Polyphosphazenes assemble antigens in virus-mimicking supramolecular constructs. • Mechanism of action reviewed in context of molecular antigen-adjuvant interactions. • Compendium of clinical and in vivo data presented for PCPP and PCEP adjuvants. • Synthetic, formulation, and biophysical characterization methodologies are reviewed. [ABSTRACT FROM AUTHOR]

At the forefront of the battle against pathogens or any endogenously released molecules, toll‐like receptors (TLRs) play an important role as the most noble pattern recognition receptors. The ability of these receptors in distinguishing "self" and "non‐self" antigens is a cornerstone in the innate immunity system; however, misregulation links inflammatory responses to the development of human cancers. It has been known for some time that aberrant expression and regulation of TLRs not only endows cancer cells an opportunity to escape from the immune system but also supports them through enhancing proliferation and angiogenesis. Over the past decades, cancer research studies have witnessed a number of preclinical and clinical breakthroughs in the field of TLR modulators and some of the agents have exceptionally performed well in advanced clinical trials. In the present review, we have provided a comprehensive review of different TLR agonists and antagonists and discuss their limitations, toxicities, and challenges to outline their future incorporation in cancer treatment strategies. [ABSTRACT FROM AUTHOR]

Infection with human papillomavirus (HPV) typically leads to cervical cancer, skin related cancers and many other tumors. HPV is mainly responsible for evading immune tumor monitoring in HPV related cancers. Toll like receptors (TLRs) are particular pattern recognition molecules. When the body is facing immune danger, it can lead to innate and direct adaptive immunity. TLR plays an important role in initiating antiviral immune responses. HPV can affect the expression level of TLR and interfere with TLR related signaling pathways, resulting in sustained viral infection and even carcinogenesis. This paper introduces the HPV virus and HPV related cancers. We discussed the present comprehension of TLR, its expression and signaling, as well as its role in HPV infection. We also provided a detailed introduction to immunotherapy methods for HPV related diseases based on TLR agonists. This will provide insights into methods that support the therapeutic method of HPV related conditions with TLR agonists., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Qi, Yang, Li, Yu and Deng.)

Self-immolative (SI) spacers are degradable chemical connectors widely used in prodrugs and drug conjugates to release pharmaceutical ingredients in response to specific stimuli. Amine-carbamate SI spacers are particularly versatile, as they have been used to release different hydroxy cargos, ranging from 2° and 3° alcohols to phenols and oximes. In this work, we describe the ability of three amine-carbamate SI spacers to release three structurally similar imidazoquinoline payloads, bearing either a 1°, a 2° or a 3° alcohol as the leaving group. While the spacers showed comparable efficacy at releasing the 2° and 3° alcohols, the liberation of the 1° alcohol was much slower, unveiling a counterintuitive trend in nucleophilic acyl substitutions. The release of the 1° alcohol payload was only possible using a SI spacer bearing a pyrrolidine ring and a tertiary amine handle, which opens the way to future applications in drug delivery systems., (© 2024 Wiley‐VCH GmbH.)

Particular interest to harness the innate immune system for cancer immunotherapy is fueled by limitations of immune checkpoint blockade. Plasmacytoid dendritic cells (pDC) are detected in a variety of solid tumors and correlate with poor clinical outcome. Release of type I interferons in response to toll-like-receptor (TLR)7 and TLR9 activation is the pDC hallmark. Mouse and human pDC differ substantially in their biology concerning surface marker expression and cytokine production. Here, we employed humanized mouse models (HIS) to study pDC function. We performed a comprehensive characterization of transgenic, myeloid-enhanced mouse strains (NOG-EXL and NSG-SGM3) expressing human interleukin-3 (hIL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) using identical humanization protocols. Only in HIS-NOG-EXL mice sufficient pDC infiltration was detectable. Therefore, we selected this strain for subsequent tumor studies. We analyzed pDC frequency in peripheral blood and tumors by comparing HIS-NOG-EXL with HIS-NOG mice bearing three different ovarian and breast tumors. Despite the substantially increased pDC numbers in peripheral blood of HIS-NOG-EXL mice, we detected TLR7/8 agonist responsive and thus functional pDCs only in certain tumor models independent of the mouse strain employed. However, HIS-NOG-EXL mice showed in general a superior humanization phenotype characterized by reconstitution of different myeloid subsets, NK cells and B cells producing physiologic IgG levels. Hence, we provide first evidence that the tumor milieu but not genetically introduced cytokines defines intratumoral (i.t.) frequencies of the rare pDC subset. This study provides model systems to investigate in vivo pro- and anti-tumoral human pDC functions.

Toll-like receptors (TLRs) are the largest pattern recognition receptors responsible for activating the innate and adaptive immune response against viruses through the release of inflammatory cytokines and antiviral mediators. Viruses are recognized by several TLRs, including TLR8, which is known to bind ssRNA structures. However, the similarities between TLR8 and TLR7 have obscured the distinctive characteristics of TLR8 activation and its importance in the immune system. Here we discuss the activation and regulation of TLR8 by viruses and its importance in therapeutical options such as vaccine adjuvants and antiviral stimulators.

Particular interest to harness the innate immune system for cancer immunotherapy is fueled by limitations of immune checkpoint blockade. Plasmacytoid dendritic cells (pDC) are detected in a variety of solid tumors and correlate with poor clinical outcome. Release of type I interferons in response to toll-like-receptor (TLR)7 and TLR9 activation is the pDC hallmark. Mouse and human pDC differ substantially in their biology concerning surface marker expression and cytokine production. Here, we employed humanized mouse models (HIS) to study pDC function. We performed a comprehensive characterization of transgenic, myeloid-enhanced mouse strains (NOG-EXL and NSG-SGM3) expressing human interleukin-3 (hIL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) using identical humanization protocols. Only in HIS-NOG-EXL mice sufficient pDC infiltration was detectable. Therefore, we selected this strain for subsequent tumor studies. We analyzed pDC frequency in peripheral blood and tumors by comparing HIS-NOG-EXL with HIS-NOG mice bearing three different ovarian and breast tumors. Despite the substantially increased pDC numbers in peripheral blood of HIS-NOG-EXL mice, we detected TLR7/8 agonist responsive and thus functional pDCs only in certain tumor models independent of the mouse strain employed. However, HIS-NOG-EXL mice showed in general a superior humanization phenotype characterized by reconstitution of different myeloid subsets, NK cells and B cells producing physiologic IgG levels. Hence, we provide first evidence that the tumor milieu but not genetically introduced cytokines defines intratumoral (i.t.) frequencies of the rare pDC subset. This study provides model systems to investigate in vivo pro- and anti-tumoral human pDC functions. [ABSTRACT FROM AUTHOR]

Mature dendritic cells (DCs) represent cellular adjuvants for optimal antigen presentation in cancer vaccines. Recently, a combination of prostaglandin E2 (PGE2) with Toll-like receptor agonists (TLR-P) was proposed as a new standard to generate superior cytokine-producing DCs with high migratory capacity. Here, we compare TLR-P DCs with conventional DCs matured only with the proinflammatory cytokines TNFα and IL-1ß (CDCs), focussing on the interaction of resulting DCs with CD8+ T-cells. TLR-P matured DCs showed elevated expression of activation markers such as CD80 and CD83 compared to CDCs, together with a significantly higher migration capacity. Secretion of IL-6, IL-8, IL-10, and IL-12 was highest after 16 h in TLR-P DCs, and only TLR-P DCs secreted active IL-12p70. TLR-P DCs as well as CDCs successfully primed multifunctional CD8+ T-cells from naïve precursors specific for the peptide antigens Melan-A, NLGN4X, and PTP with comparable priming efficacy and T-cell receptor avidity. CD8+ T-cells primed by TLR-P DCs showed significantly elevated expression of the integrin VLA-4 and a trend for higher T-cell numbers after expansion. In contrast, TLR-P DCs displayed a substantially reduced capability to cross-present CMVpp65 protein antigen to pp65-specific T cells, an effect that was dose-dependent on PGE2 during DC maturation and reproducible with several responder T-cell lines. In conclusion, TLR-P matured DCs might be optimal presenters of antigens not requiring processing such as short peptides. However, PGE2 seems less favorable for maturation of DCs intended to process and cross-present more complex vaccine antigens such as lysates, proteins or long peptides. [ABSTRACT FROM AUTHOR]

Based on experimental results of a mouse model provided in the literature, we develop a mathematical model by using system biology approach, aiming to investigate immunotherapy for 4T1 breast cancer. It is worth to mention that only 4 types of cells (tumor cells, CD8 + T cells, regular T cells (Tregs), and tumoricidal myeloid CD11b + Gr1dim cells) are quantitatively measured in experiments, which make the immunotherapy modelling more difficult since only limited system knowledge is available. To overcome the difficulty, the mathematical model is proposed by employing Evolutionary Computation to optimize the system parameters. Furthermore, with the mathematical model, analysis can be conducted to capture the inherent properties of the model, such as the number and stability of equilibria, and parameter sensitivity analysis, which disclose the nature of 4T1 breast cancer from a system biological perspective. Not limited to replication of experimental results, we further show that the mathematical model is in fact a prognostic immunotherapy model that can predict treatment outcomes of various cases; for instance, different combinations of drug delivery schedules. By virtue of computational convenience, it is relatively easy to intensively investigate most of the treatments that are impossible for animal models or clinical trials. In other words, a mathematical model based on system biology can provide meaningful reference when exploiting more effective treatment protocols. [ABSTRACT FROM AUTHOR]

Agonists for toll-like receptors (TLRs) have shown promising activities against cancer. In the present study, a squalene-based nanoemulsion (NE) was loaded with resiquimod, a TLR7/8 agonist for therapeutic delivery. R848 NE was developed and characterized for long-term stability. In vitro and in vivo antitumor immunity of R848 NE were also evaluated in combination with SD-101, a CpG-containing TLR9 agonist. In vitro studies demonstrated strong long-term stability and immune responses to R848 NE. When combined with SD-101, strong antitumor activity was observed in MC38 murine colon carcinoma model with over 80% tumor growth inhibition. The combination treatment showed a 4-fold increase in systemic TNFa production and a 2.6-fold increase in Cd8a expression in tumor tissues, suggesting strong cell-mediated immune responses against the tumor. The treatment not only demonstrated a strong antitumor immunity by TLR7/8 and TLR9 activations but also induced PD-L1 upregulation in tumors, suggesting a potential therapeutic synergy with immune checkpoint inhibitors.

Dendritic cells (DCs), and especially so conventional type I DCs (cDC1s), are fundamental regulators of anticancer immunity, largely reflecting their superior ability to engulf tumor-derived material and process it for cross-presentation on MHC Class I molecules to CD8 + cytotoxic T lymphocytes (CTLs). Thus, investigating key DC functions including (but not limited to) phagocytic capacity, expression of CTL-activating ligands on the cell surface, and cross-presentation efficacy is an important component of multiple immuno-oncology studies. Unfortunately, DCs are terminally differentiated cells, implying that they cannot be propagated indefinitely in vitro and hence must be generated ad hoc from circulating or bone marrow-derived precursors, which presents several limitations. Here, we propose a simple, cytofluorometric method to quantify phenotypic activation markers including CD80, CD86 and MHC class II molecules on the surface of a conditionally immortalized immature DC line that can be indefinitely propagated in vitro but also driven into maturation at will with a simple change in culture conditions. Upon appropriate scaling and automatization, this approach is compatible with high-throughput screening programs for the discovery of novel DC activators that do not suffer from batch variability and other limitations associated with the generation of fresh DCs., Competing Interests: Competing interests O.K. is a scientific cofounder of Samsara Therapeutics. G.K. is on the Board of Directors of the Bristol Myers Squibb Foundation France. G.K. is a scientific cofounder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. G.K. is in the scientific advisory boards of Hevolution, Institut Servier, Longevity Vision Funds and Rejuveron Life Sciences. G.K. is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. G.K.'s wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Merus, Transgene, 9m, Tusk and Roche, was on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. G.K.'s brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. The funders had no role in the design of the study; in the writing of the manuscript, or in the decision to publish the results. LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options. The other authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans. [ABSTRACT FROM AUTHOR]

The magnitude of immune responses to vaccination is a critical factor in determining protection from disease. It is known that cigarette smoke dampens the immune system and increases the risk of vaccine-preventable diseases. We reported that nicotine, the immunosuppressive component of cigarette smoke, disrupts the differentiation and functional properties of DC, which are pivotal in the initiation of immune response to vaccines. We also reported that TLR agonists act in synergy and boost DC maturation, DC-NK crosstalk and ultimately naïve T cell polarization into effector Th1 and Tc1 cells. Here, we investigated whether the combination of TLR agonists could diminish the degrading effects of nicotine on DC-NK mediated effector T cell generation. We found that none of TLR agonists, single or combined, were able to diminish completely the adverse effects of nicotine on DC. However, TLR3, TLR4, and TLR8 agonists acted as the most effective adjuvants to increase the expression levels of antigen-presenting, costimulatory molecules and production of cytokines by nicotine-exposed DC (nicDC). When combined, TLR3 + 8 and TLR4 + 8 synergistically optimized nicDC maturation and IFN-γ secretion from nicotine-exposed NK (nicNK) during co-cultures. Interestingly, in contrast to DC-NK-T, co-cultures of nicDC-nicNK-T treated with TLR3 + 8 or TLR4 + 8 agonists produced a similar frequency of effector memory Th1 and Tc1 cells. However, the effector cells from TLR4 + 8 followed by TLR3 + 8 treated nicDC-nicNK-T co-cultures produced significantly more IFN-γ when compared with aluminum salt treated co-culture. Our data suggest that addition of appropriate TLR agonists to vaccine formulation could potentially augment the immune response to vaccination in smokers. [ABSTRACT FROM AUTHOR]

Immunotherapy emerges as a fundamental approach in cancer treatment. Up to date, the efficacy of numerous different immunotherapies has been evaluated. The use of microorganisms or their parts for immune cell activation, referred to as Pathogen-Associated Molecular Patterns (PAMPs), represents highly promising concept. The therapeutic effect of PAMPs can be further amplified by suitable combination of different types of PAMPs such as Toll like receptor (TLR) agonists and phagocytosis activating ligands. Previously, we used the combination of phagocytosis activating ligand (mannan) and mixture of TLR agonists (resiquimod (R-848), poly(I:C), inactivated Listeria monocytogenes ) for successful treatment of melanoma in murine B16-F10 model. In the present study, we optimized the composition and timing of previously used mixture. Therapeutic mixture based on well-defined chemical compounds consisted of mannan anchoring to tumor cell surface by biocompatible anchor for membranes (BAM) and TLR agonists resiquimod, poly(I:C), and lipoteichoic acid (LTA). The optimization resulted in (1) eradication of advanced stage progressive melanoma in 83% of mice, (2) acquisition of resistance to tumor re-transplantation, and (3) potential anti-metastatic effect. After further investigation of mechanisms, underlying anti-tumor responses, we concluded that both innate and adaptive immunity are activated and involved in these processes. We tested the efficacy of our treatment in Panc02 murine model of aggressive pancreatic tumor as well. Simultaneous application of agonistic anti-CD40 antibody was necessary to achieve effective therapeutic response (80% recovery) in this model. Our results suggest that herein presented immunotherapeutic approach is a promising cancer treatment strategy with the ability to eradicate not only primary tumors but also metastases. [ABSTRACT FROM AUTHOR]

Background Most current prophylactic vaccines confer protection primarily through humoral immunity. Indeed, aluminum salts which have been widely used as adjuvants in vaccines primarily enhance Th2-driven antibody responses. Therefore, new vaccines formulation is moving toward a careful selection of adjuvants that also elicit significant Th1 or Tc1 responses. Several TLR agonists have been tested as potential new adjuvants in clinical and preclinical studies with some efficacy. These studies suggest that combining more than one of TLR ligands enhances the magnitude of immune responses to cancer and infectious disease. Objectives In order to evaluate the synergistic effect of TLR agonists for effective induction of cellular immunity, we investigated the effects of single and/or combined TLR agonists on monocyte-derived DC maturation, DC-NK crosstalk and ultimately naïve T cells polarization into effector T cells. Results Among the adjuvants tested, we found that TLR3, TLR4, TLR7/8 and TLR8 agonists were the most effective adjuvants to increase the expression levels of antigen-presenting, co-stimulatory molecules and production of cytokines by maturing DCs. When combined, TLR3 + 8 and TLR4 + 8 synergistically optimized DC maturation and IFN-γ secretion from NK cells co-cultured with DCs. Interestingly, co-culture of DC-NK-T treated with aluminum salt produced the highest percentage of effector memory CFSE-CCR7- Th1 cells whereas TLR3 + 8 and TLR4 + 8 treated co-cultures produced the highest percentage of effector memory CFSE-CCR7- Tc1 cells producing IFN-γ. Finally, while both TLR3 + 8 or TLR4 + 8 treated co-cultures generated similar frequency of Th1 and Tc1 effector cells, the effector cells from the latter co-culture produced quantitatively more IFN-γ in the supernatant. Conclusion Our data indicate that if in need of an enhanced DC-NK mediated cellular immunity one may select TLR agonists with defined synergistic effects. [ABSTRACT FROM AUTHOR]