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Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of TLR agonists to antigenic proteins offers a unique approach to co-deliver both constituents to the same cell-enhancing vaccine efficacy while minimizing reactogenicity. However, the paucity of simple and effective linker chemistries continues to hamper progress. Here, we present a modular, PEG-based linker system compatible with even extremely lipophilic and challenging TLR7/8 agonists. To advance the field and address previous obstacles, we offer the most straightforward and antigen-preserving linker system to date. These antigen-adjuvant conjugates enhance antigen-specific immune responses in mice, demonstrating the power of our approach within the context of modern vaccinology.

CD8+-T-Effektor-Zellen spielen eine Schlusselrolle in der Identifikation und Elimination von Tumorzellen. Tumoren entkommen einer effektiven T‑Zell-Antwort durch Induktion eines Erschopfungszustands, der die zytotoxische Kapazitat der Effektorzellen mindert. Neuartige Checkpointinhibitoren wirken u. a. durch Reaktivierung erschopfter, dysfunktionaler T‑Zellen. CD8+-T-Zellen eliminieren Tumorzellen nach Darbietung tumorspezifischer Antigene durch antigenprasentierende Zellen (APZ). Die APZ-vermittelte Tumorerkennung wird v. a. Toll-like-Rezeptoren (TLR) stimuliert. Untersucht wurde der Einfluss von TLR-Agonisten auf APZ sowie stimulierende und inhibierende Signalwege der T‑Zell- und APZ-Interaktion. Die Genexpression von Interleukin (IL-)12 und PDL1 wurde nach Stimulation von CD14+-Zellen mit CpG uber 0, 8, 24 und 48 h analysiert. Die Expression des inhibitorischen kappa-B-Proteins (IκBα) nach Stimulation mit CpG wurde mittels Westernblot ausgewertet. CD8+-T-Zellen wurden uber 72 h mit und ohne PD1-Checkpointblockade stimuliert und mittels Durchflusszytometrie auf die Expression von PD1, Tim‑3, CTLA4 und Lag3 untersucht. Durch TLR-Stimulation (unmethylierte CpG-DNA) von APZ wurden immunstimulierende Signale wie die IL-12-Expression gesteigert, aber auch immuninhibierende Signalwege wie die PDL1-Expression aktiviert. Diese Signalwege werden NF-κB-vermittelt. Nach alleiniger Blockade der PD1-PDL1-Signalwege zeigte sich eine Uberexpression anderer Immuncheckpointrezeptoren als potenzielle Ursache fur fehlendes Ansprechen auf TLR-Stimulation mit PD1-Checkpointblockade. Durch eine TLR-Stimulation exprimieren APZ im Tumormikromilieu NF-κB-vermittelt PDL1 und tragen so zur Erschopfung von CD8+-T-Zellen bei. Der Effekt einer PD1-Blockade wird dabei potenziell durch Uberexpression anderer Checkpointinhibitoren beeintrachtigt.

Therapies based on Toll Like Receptor agonists (TLRa) are emerging as a promising modality for cancer immunotherapy to recruit antitumor T-cells in unresponsive immunologically “cold” tumors. Often, combinations of agonists are employed to synergistically enhance efficacy. However, low efficacy and severe toxicities deter these TLR-based therapeutics from further clinical applications. Studies have suggested that the rapid systemic diffusion of agonists to nontarget tissues is the primary cause. To address this challenge, we developed supramolecular nanotherapeutics of covalently linked TLRas for multivalent, synergistic interactions by drawing inspiration from immune recognition of pathogens. This new nanotherapeutic increased stimulation of key pro-inflammatory cytokines and remarkably enhanced CD8 and NK cell-mediated antitumor response while exhibiting ultralow off-target toxicity in an aggressive B16.F10 tumor model. Results from our studies thereby indicate that such supramolecular immune-agonist therapeutics may be further developed as a viable treatment modality for cancer immunotherapy., Therapies based on Toll Like Receptor agonists (TLRa) are a promising cancer immunotherapy, and our studies indicate that supramolecular TLRa heterodimer therapeutics may be developed as an efficacious and less toxic treatment.

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[Formula: see text] T cells, regular T cells (Tregs), and tumoricidal myeloid CD11b[Formula: see text]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.

The development of vaccines has resulted in a dramatic decrease in the number of cases of diseases, such as measles and smallpox. With the emergence of Ebola and the Zika virus, there is a greater need for the development of more effective and safer vaccines. However, the challenge with designing new immunotherapies is that little is still known about how vaccines work, since most have been empirically determined. Thus, our group is interested in using chemical tools to probe and understand the immune response with the goal of designing more effective vaccines. Dendritic cells, a vital part of the innate immune system, contain Toll-like receptors (TLRs) that are activated by components of pathogens, such as bacterial oligonucleotides and lipopeptides. These molecules are immune agonists that can act as adjuvants, which help elicit or enhance an immune response toward a non-immunogenic protein antigen, and are commonly used in vaccines. Recent studies indicate that vaccines containing multiple TLR agonists enhance the immune response toward a target pathogen compared to the use of a single ligand. A prime example is the Yellow Fever Vaccine, one of the most successful vaccines, which activates the immune system through four different TLRs. Due to this precedence, we hypothesized that activating specific receptors in a precise spatial manner could modulate how the immune system responds by mimicking natural pathogens and therefore control downstream pathways. My research focuses on synthesizing multi-TLR agonist conjugates to study the spatial organization of multiple TLR agonists and how different combinations of agonists affect the immune response, as observed by cellular and antibody responses. To study the effect of multiple TLR agonists on the immune response, we chemically modified whole cell antigens with different TLR agonists as well as covalently conjugated multiple TLR agonists together to present them in a localized manner. As a result of chemical modification and linkage, we observed distinct changes in immune activation, via cytokine production and antibody responses, suggesting implications for downstream immune system signaling. We are applying our findings to more rationally develop safer and more effective vaccine adjuvants and immunotherapies.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immunostimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll like receptor (TLR) 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell-dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally-related adjuvant AS01(B) in mice, and primed robust germinal center B cell, T(FH), and HIV tier 2 neutralizing antibodies in non-human primates. Altogether, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.

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.

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.

Chronic hepatitis B virus (HBV) infection remains a major global health problem. The immunopathology of the disease, especially the interplay between HBV and host innate immunity, is poorly understood. Moreover, inconsistent literature on HBV and host innate immunity has led to controversies. However, recently, there has been an increase in the number of studies that have highlighted the link between innate immune responses, including Toll-like receptors (TLRs), and chronic HBV infection. TLRs are the key sensing molecules that detect pathogen-associated molecular patterns and regulate the induction of pro- and anti-inflammatory cytokines, thereby shaping the adaptive immunity. The suppression of TLR response has been reported in patients with chronic hepatitis B (CHB), as well as in other models, including tree shrews, suggesting an association of TLR response in HBV chronicity. Additionally, TLR agonists have been reported to improve the host innate immune response against HBV infection, highlighting the potential of these agonists as immunomodulators for enhancing CHB treatment. In this study, we discuss the current understanding of host innate immune responses during HBV infection, particularly focusing on the TLR response and TLR agonists as immunomodulators.

Small-molecule modulators of TLR8 have drawn much interests as it plays pivotal roles in the innate immune response to single-stranded RNAs (ssRNAs) derived from viruses. However, their clinical uses are limited because they can invoke an uncontrolled, global inflammatory response. The efforts described herein culminate in the fortuitous discovery of a tetrasubstituted imidazole CU-CPD107 which inhibits R848-induced TLR8 signaling. In stark contrast, CU-CPD107 shows unexpected synergistic agonist activities in the presence of ssRNA, while CU-CPD107 alone is unable to influence TLR8 signaling. CU-CPD107’s unique, dichotomous behavior sheds light on a way to approach TLR agonists. CU-CPD107 offers the opportunity to avoid the undesired, global inflammation side effects that have rendered imidazoquinolines clinically irrelevant, providing an insight for the development of antiviral drugs., Toll-like receptor 8 (TLR8) plays essential roles in the innate immune response to viral single-stranded RNA (ssRNA), so small molecule modulators of TLR8 are of interest, however adverse effects limit their use. Here, the authors report a tetrasubstituted imidazole CU-CPD107 with dichotomous behaviour, which inhibits R848-induced TLR8 signaling, but shows synergistic activity in the presence of ssRNA, making it a potential antiviral agent.

A key challenge facing immunotherapy is poor infiltration of T cells into tumors, along with suppression of cells reaching these sites. However, macrophages make up a majority of immune cell infiltrates into tumors, creating natural targets for immunotherapies able to direct macrophages away from tumor-supportive functions and toward anti-tumor phenotypes. Recent studies demonstrate that toll-like receptors (TLRs) - pathways that quickly trigger early immune responses - play an important role in polarizing macrophages. Here, we present emerging ways in which TLR signaling is being manipulated in macrophages to create new opportunities for cancer immunotherapy. In particular, we discuss approaches to deliver TLR agonists, to leverage biomaterials in these therapies, and to couple TLR-based approaches with other frontline treatments as combination cancer therapies.

Antiviral research has focused mainly on viral targets. However, cellular targets involved in the viral life cycle and antiviral response are becoming more attractive for research, providing a variety of opportunities for antiviral therapy. Toll-like receptors (TLR) play an important role in activation of both innate and adaptive immune systems, including a response to respiratory viral infections. In this review we shall discuss TLRs as potential targets for development of novel antiviral drugs including the mechanisms for induction the antiviral response by means of type I interferon production, as well as viral evasion strategies. In addition, we describe several new molecules that have been applied as TLR agonists or antagonists. The safety issues are also discussed.

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) has caused infection in different parts of the world, especially in Africa, the Middle East, and the Mediterranean. HHV8 is a gamma-2 class herpesvirus and its genome contains 26 open reading frames. K1 is a highly variable glycoprotein and can be used as a vaccine candidate. The aim of this study was to design a new vaccine by using immunoinformatics analysis and adding approved adjuvants. In this study, several computational programs were employed to define the most capable region in K1-based immunologic properties; also, different possible adjuvants and universal T-helper agonists were added to the new construct. The construct was examined to define the 3D structure, physicochemical properties and immunologic features. In addition, docking analysis was done to define the ability of the new construct to attach to mTLR. A region (155–170) was selected to be used in the new proposed vaccine; in addition, two TLR agonist adjuvants and two universal T-helpers were added to boost the immune response. Analysis of the new construct showed that the designed protein was able to be used as a vaccine and be expressed in the host. Moreover, the high energy value in docking analysis showed the high ability of this vaccine to induce immune system. The designed vaccine is expected to be capable of generating humoral and cellular responses that are crucial to protect against HHV8 as well as the vaccine has appropriate physicochemical properties and acceptable stability in different host cells.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(L-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.

Immunostimulatory agents such as Toll-like receptor (TLR) agonists have shown promising antitumor efficacy but are associated with therapy-related toxicities when delivered systemically. Immune-stimulating antibody conjugates are now shown to deliver TLR agonists with potent preclinical antitumor activities.

Vaccine development utilizing various platforms is one of the strategies that has been proposed to address the coronavirus disease 2019 (COVID-19) pandemic. Adjuvants are critical components of both subunit and certain inactivated vaccines because they induce specific immune responses that are more robust and long-lasting. A review of the history of coronavirus vaccine development demonstrates that only a few adjuvants, including aluminum salts, emulsions, and TLR agonists, have been formulated for the severe acute respiratory syndrome-associated coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and currently the SARS-CoV-2 vaccines in experimental and pre-clinical studies. However, there is still a lack of evidence regarding the effects of the adjuvants tested in coronavirus vaccines. This paper presents an overview of adjuvants that have been formulated in reported coronavirus vaccine studies, which should assist with the design and selection of adjuvants with optimal efficacy and safety profiles for COVID-19 vaccines.

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.

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.

This master's thesis studies the efficacy of cancer immunotherapy based on TLR agonists and ligands stimulating phagocytosis, abbreviated as MBTA therapy, using a mouse model of pancreatic adenocarcinoma. The main goals are: (1) to enhance the efficacy of MBTA in the case of large tumors, (2) to enhance the efficacy of MBTA using a bilateral pancreatic adenocarcinoma mouse model, (3) to examine the potential of lipoteichoic acid to opsonize cancer cells and stimulate phagocytic cells.

In a continuing effort to discover novel TLR agonists, herein we report on the discovery and structure-activity relationship of novel tetrahydropyridopyrimidine TLR 7/8 agonists. Optimization of this series towards dual agonist activity and a high clearance profile resulted in the identification of compound 52a1. Evaluation in vivo revealed an interferon stimulated response (ISG) in mice with limited systemic exposure and demonstrated the potential in antiviral treatment or as a vaccine adjuvant.

Interleukin 12 (IL-12) is a potent proinflammatory cytokine that plays a central role in regulating both innate and adaptive immune responses. This cytokine has been tested as a cancer immunotherapy agent based on its impressive anti-tumor effects in animal models. Clinically, systemic delivery of IL-12 incurred dose-limiting toxicities because of its pharmaco-kinetic extremes of peaks and troughs, a result of its short serum half-life. Here, we evaluated mDF6006, a mouse IL-12 Fc-fusion protein which was designed to have a prolonged half-life with equivalent potency to recombinant mouse IL-12. We hypothesized that local intra-tumoral (IT) delivery of low doses IL-12Fc would lead to immune-therapeutic potential. To screen candidates for in situ vaccination, we have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting immune response is detected by the regression of the distant, untreated tumor. Using this assay for abscopal therapeutic effects we have tested a variety of immune stimulatory ligands and antibodies against immune checkpoints and T cell co-stimulatory targets, alone and in multiple combinations. Among these, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG), a TLR9 ligand, and IL-12Fc has provided the most impressive results. TLRs, expressed in antigen-presenting cells and are components of the innate immune system that recognize molecular patterns on bacterial, fungal, or viral pathogens. In a mouse model of lymphoma, IT injection of CpG and IL-12Fc, combined to cause tumor eradication at both the injected site and the untreated tumor. All these results were dependent on T cells in the animal. During this therapy we observed a significant increase in activated B cells within the treated tumor and especially in its draining lymph nodes (dLN). This B cell stimulation was contributed by both the CpG and the IL12-Fc. Surprisingly, depletion of the animals of B cells by an anti-CD20 antibody completely ablated the abscopal therapeutic effect. B cells function as antigen-presenting cells, secrete cytokines and are precursors of antibody producing plasma cells. To evaluate which of these roles B cells play in the therapeutic efficacy of CpG and IL-12Fc we isolated B cells from the LN of the treated mice and co-cultured them with T cells. These B cells activated T cells in an antigen-specific manner that was MHC dependent. No such T cell activation occurred when MHC molecules were blocked with anti-MHC antibodies or when the cells were physically separated by a semi-permeable membrane. Thus, local CpG and IL12-Fc directly activated B cells to present tumor antigens to specific T cells in the tumor microenvironment, resulting ultimately in an abscopal tumor response. IL-12Fc and CpG are both currently in phase-1/2 trials as single agents and in combinations with checkpoint antibodies. Our results provide strong rationale for using low doses of IL-12Fc with CpG as an in situ therapeutic vaccine for lymphoma. Disclosures Levy: Virsti: Membership on an entity's Board of Directors or advisory committees; Khloris: Membership on an entity's Board of Directors or advisory committees; Abintus Bio: Membership on an entity's Board of Directors or advisory committees; Kira: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees.

BackgroundT-cell checkpoint receptors are expressed when T-cell are activated, and activation of these receptors can impair the function of T-cells and their anti-tumor efficacy.1 We previously found that T-cells activated with cognate antigen increase the expression of PD-1, while this can be attenuated by the presence of specific Toll-like receptor (TLR) agonists.2 3 This effect was mediated by IL-12 secretion from professional antigen presenting cells and resulted in CD8+ T cells with greater anti-tumor activity. In the current report, we sought to determine whether combination of TLR agonists can further affect the expression of T-cell checkpoint receptors and improve T-cell anti-tumor immunity.MethodsOT-1 CD8+ T cells were stimulated with peptide (SIINFEKL) and dendritic cells (DC) in the presence of two different TLR agonists. The cells were collected and evaluated for the expression of T-cell checkpoint receptors (PD-1, CTLA-4, CD160, CD244, LAG-3, TIM-3, TIGIT and VISTA) by flow cytometry, and for transcriptional changes by RNA-seq. Purified DC were stimulated with TLR combinations and evaluated for cytokine release by ELISA. The anti-tumor efficacy of vaccination using peptide and TLR agonist combinations was evaluated in EG7-OVA tumor-bearing mice.ResultsActivation of CD8+ T cells in the presence of specific TLR ligands resulted in decreases in expression of PD-1 and/or CD160. These changes in T-cell checkpoint receptor expression were modestly affected when TLR ligands were used in combination, and notably with combinations of TLR1/2, TLR3, and TLR9 agonists. Immunization of tumor-bearing mice, co-administered with combinations of these agonists, showed greater anti-tumor effects. However, while the effect of TLR1/2 and/or TLR9 was abrogated in IL12KO mice, TLR3 demonstrated anti-tumor activity when co-administered with peptide vaccine. RNA sequencing of TLR-conditioned CD8+ T-cells revealed IL-12 pathway activation, and IFNß pathway activation following TLR3 stimulation. Stimulation of DC with TLR3 agonist, alone or in combination with other TLR agonists, resulted in increased IL-12 and IFNß secretion. Co-incubation of OT-1 splenocytes with rIL12 and/or rIFNß during peptide activation led to reduced expression of PD-1, and this could be reversed with antibodies blocking IL12R or IFNAR-1.ConclusionsMultiple TLR agonists can modulate the expression of T-cell checkpoint receptors, notably PD-1, by upregulating the secretion of IL-12 and IFNß. These data provide the mechanistic rationale for choosing optimal combinations of TLR ligands to use as adjuvants to improve the efficacy of anti-tumor vaccines.ReferencesJin H-T, et al. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proceedings of the National Academy of Sciences 2010;107(33):14733–14738.Zahm CD, Colluru VT, McNeel DG. Vaccination with high-affinity epitopes impairs antitumor efficacy by increasing PD-1 expression on CD8+ T cells. Cancer Immunology Research 2017;5(8):630–641.Zahm CD, et al. TLR stimulation during T-cell activation lowers PD-1 expression on CD8+ T Cells. Cancer Immunology Research 2018;6(11):1364–1374.