TLR Agonists Research Articles

Inflammatory lung injury is associated with endothelial cell mitochondrial fission and requires the nitration of RhoA and cytoskeletal remodeling

Higher levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a TLR4 agonist, are associated with poor clinical outcomes in sepsis-induced acute lung injury (ALI). Little is known regarding the mechanisms by which eNAMPT is involved in ALI. Our recent work has identified a crucial role for mitochondrial dysfunction in ALI. Thus, this study aimed to determine if eNAMPT-mediated inflammatory injury is associated with the loss of mitochondrial function. Our data show that eNAMPT disrupted mitochondrial bioenergetics. This was associated with cytoskeleton remodeling and the loss of endothelial barrier integrity. These changes were associated with enhanced mitochondrial fission and blocked when Rho-kinase (ROCK) was inhibited. The increases in mitochondrial fission were also associated with the nitration-mediated activation of the small GTPase activator of ROCK, RhoA. Blocking RhoA nitration decreased eNAMPT-mediated mitochondrial fission and endothelial barrier dysfunction. The increase in fission was linked to a RhoA-ROCK mediated increase in Drp1 (dynamin-related protein 1) at serine(S)616. Another TLR4 agonist, lipopolysaccharide (LPS), also increased mitochondrial fission in a Drp1 and RhoA-ROCK-dependent manner. To validate our findings in vivo, we challenged C57BL/6 mice with eNAMPT in the presence and absence of the Drp1 inhibitor, Mdivi-1. Mdivi-1 treatment protected against eNAMPT-induced lung inflammation, edema, and lung injury. These studies demonstrate that mitochondrial fission-dependent disruption of mitochondrial function is essential in TLR4-mediated inflammatory lung injury and identify a key role for RhoA-ROCK signaling. Reducing mitochondrial fission could be a potential therapeutic strategy to improve ARDS outcomes.

Selective Timing of Maternal Immune Activation Alters Developmental Milestones, Behavior, and Blood-Brain Barrier Integrity

Background and Hypothesis: Maternal immune activation (MIA) has been associated with increased risk for neuropsychiatric disease. Fetal exposure to maternal infection activates different toll-like receptors (TLRs) to initiate innate inflammatory responses in mother and fetus. The goal of the current study is to determine sex-dependent aspects of MIA during mid-gestation on neurodevelopment. Methods: Timed-pregnant female mice were administered RQ (TLR7 agonist) or vehicle saline on embryonic day (E) 12.5. Maternal and fetal cytokines were measured to ensure infection of pregnant dam (E15). Offspring were assessed postnatally for developmental milestones and behavior. Adult offspring were perfused with fluorescein isothiocyanate (FITC; 100mg/mL) to visualize blood vessel integrity (FITC leakage; Frahm & Tobet, 2015). To determine neural molecular mechanisms for behavioral changes, sections through the paraventricular nucleus of the hypothalamus (PVN) of offspring were immunolabeled for Glial Fibrillary Acidic Protein (GFAP; astrocytic end feet), Desmin (pericytes) and IBA-1 (microglia). Results: Maternal cytokines IL-6 (p<0.05) and IL-10 (p<0.01) were higher while TNFα (p<0.01) and IL-17 were lower 3 days after RQ-exposure. In fetuses (E15), IL-6 (p<0.05) and IL-17 (p<0.0001) were greater, while IL-10 was higher only in males (p<0.001) exposed to maternal infection. RQ-exposed males also had reduced TNFα (p<0.05). Additionally, RQ-exposed offspring had lower body weights at birth and delayed eye openings. Females exposed to maternal RQ exhibited slower onset of puberty with delayed vaginal openings. On the behavioral side, juvenile and adult offspring of RQ injected moms exhibited less social-like behavior (p<0.001 for all groups) with lower hedonic-like behavior selectively in females (p<0.001). RQ-offspring further showed greater leakage in the PVN indicated by more FITC in the extravascular space (males p<0.001, females p<0.01). GFAP + (astrocytic end feet) coverage of FITC-labeled vessels was higher in the PVN selectively in RQ males (p<0.001). Desmin+ (pericyte) coverage was greater in the PVN of RQ males (p<0.01) and females (p<0.001). Finally, there was greater number of IBA-1+ cells in the adult offspring PVN of both sexes (p<0.01) after maternal injection of RQ. Conclusions: This study provides support for sex-dependent influences of fetal antecedents for altered brain development and behavioral outputs that could be indicative of increased susceptibility for adult disorders through immune mechanisms. Future studies will examine how timing of infection during gestation (mid vs. late) changes neurodevelopmental outputs in offspring. Supported by ORWH-NIMH U54 SCORE-MH118919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A novel interaction between complement product C4a and the receptor for advanced glycation end products (RAGE)

Abstract Complement proteins (C) including their split products play important roles in host defense and homeostasis. Anaphylatoxins C3a and C5a, by binding to their receptors on immune and non-immune cells, mediate cell recruitment and the release of proinflammatory molecules. The receptor and function of C4a remain unclear. C3a, however, in addition to its conventional receptor C3aR, is known to bind to the receptor for advanced glycation end products (RAGE), a receptor implicated in initiating and sustaining inflammatory responses. This interaction can also be mediated by the TLR9 agonist, CpG. C4a is similar to C3a in its structure and net charge. Therefore, we hypothesize that C4a may interact with RAGE. We performed co-immunoprecipitation experiment on 293T cell lysate expressing C4a and RAGE and found that RAGE did not co-immunoprecipitate with C4a, indicating that C4a does not directly interact with RAGE. Using fluorescently labelled C4a protein, we found that there was no significant C4a binding to 293T cells irrespective of RAGE overexpression by flow cytometry. However, by preincubating C4a with CpG, we detected elevated C4a binding to RAGE-expressing 293T cells compared to control cells. These findings suggest that CpG mediates interaction between C4a and RAGE. Our ongoing investigations focus on determining the binding properties and the functional impact on inflammatory response of this novel interaction.

Combining toll-like receptor agonists with immune checkpoint blockade affects antitumor vaccine efficacy

BackgroundT cell checkpoint receptors are expressed when T cells are activated, and modulation of the expression or signaling of these receptors can alter the function of T cells and their...

Effects of toll-like receptor agonists and SARS-CoV-2 antigens on interferon (IFN) expression by peripheral blood CD3+ T cells from COVID-19 patients

BackgroundSignaling by toll-like receptors (TLRs) initiates important immune responses against viral infection. The role of TLRs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not well elucidated. Thus, we investigated the interaction of TLRs agonists and SARS-COV-2 antigens with immune cells in vitro. Material & methods30 coronavirus disease 2019 (COVID-19) patients (15 severe and 15 moderate) and 10 age and sex-matched healthy control (HC) were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and activated with TLR3, 7, 8, and 9 agonists, the spike protein (SP) of SARS-CoV-2, and the receptor binding domain (RBD) of SP. Frequencies of CD3+IFN-β+ T cells, and CD3+IFN-γ+ T cells were evaluated by flow cytometry. Interferon (IFN)-β gene expression was assessed by qRT-PCR. ResultsThe frequency of CD3+IFN-β+ T cells was higher in PBMCs from moderate (p < 0.0001) and severe (p = 0.009) patients at baseline in comparison with HCs. The highest increase in the frequency of CD3+IFN-β+ T cells in cell from moderate patients was induced by TLR8 agonist and SP (p < 0.0001 for both) when compared to HC, while, the highest increase of the frequency of CD3+IFN-β+ T cells in sample of severe patients was seen with TLR8 and TLR7 agonists (both p = 0.002). The frequency of CD3+IFN-γ+ T cells was significantly increased upon stimulation with TLR agonists in cell from patients with moderate and severe COVID-19, compared with HC (all p < 0.01), except with TLR7 and TLR8 agonists. The TLR8 agonist did not significantly increase the frequency of CD3+IFN-γ+ T cells in PBMCs of severe patients, but did so in cells from patients with moderate disease (p = 0.01). Moreover, IFN-β gene expression was significantly upregulated in CD3+T cells from moderate (p < 0.0001) and severe (p = 0.002) COVID-19 patients, compared to HC after stimulation with the TLR8 agonist, while, stimulation of T cells with SP, significantly up-regulated IFN-β mRNA expression in cells from patients with moderate (p = 0.0003), but not severe disease. ConclusionStimulation of PBMCs from COVID-19 patients, especially patients with moderate disease, with TLR8 agonist and SP increased the frequency of IFN-β-producing T cells and IFN-β gene expression.

Enhanced Efficacy of a TLR3 Agonist Delivered by Cowpea Chlorotic Mottle Virus Nanoparticles

Intratumoral immunotherapies are those that are administered directly into a tumor to remodel the local tumor microenvironment and stimulate systemic anti‐tumor immunity. Small molecule toll‐like receptor (TLR) agonists are undergoing development as intratumoral immunotherapies, and here the TLR3 agonist poly(I:C) is considered. Because small molecule therapeutics often suffer rapid washout effects and ineffective immune cell uptake, poly(I:C) is encapsulated into nanoparticles derived from cowpea chlorotic mottle virus (CCMV). The particles (but not the separate components) stimulate the activity of macrophages in vitro and are able to reduce tumor growth and prolong survival in mouse models of colon cancer and melanoma. CCMV‐poly(I:C) is also combined with oxaliplatin and found the combination therapy to be even more potent, strongly inhibiting tumor growth and increasing survival. The analysis of immune markers reveals that CCMV‐poly(I:C) VLPs with oxaliplatin promoted the infiltration and activation of CD4+ and CD8+ cells and the production of IL‐4 and IFN‐γ, indicating a synergistic immunogenic effect. The combined treatment also enhances the rate of apoptosis and immunogenic cell death (ICD). The data support the development of combination therapies involving immunomodulatory plant virus nanoparticles and antineoplastic drugs to attack tumors directly and via the activation of innate and adaptive immune responses.

Advax-SM™-Adjuvanted COBRA (H1/H3) Hemagglutinin Influenza Vaccines.

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

P179 CD19hi B cells express T-bet and display activated metabolism and function in rheumatoid arthritis and systemic lupus erythematosus patient samples

Abstract Background/Aims CD19, a co-receptor of the B cell receptor, fine-tunes the threshold for B-cell activation with implications for cell metabolism and function. Activating receptors such as CD40, IFNγR and TLRs also influence B cell activation. B cell subpopulation, double negative (DN, IgD-CD27-) T-bet+ B cells, is considered pathogenic in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Here, we explored whether CD19hi B cells differed from CD19lo B cells with regards to proliferation, metabolism, and function. Methods Peripheral blood samples from nine healthy participants, 16 RA and 20 SLE patients were analysed. Subgroup analysis of rituximab-treated (RTX-T), n = 6 versus rituximab naïve (RTX-N), n = 6, was performed. B cells or peripheral blood mononuclear cells were stimulated with anti-IgG, anti-IgM and anti-human CD40 antibodies and a TLR9 agonist for 20 hours prior to flow cytometric analysis of B cell expression of phosphorylated AMPK (pAMPK, the master regulator of metabolism), intracellular expression of Ki67 (proliferation marker), and cytokines IL-6 and IL-10. Statistical analysis was performed using GraphPad Prism v10.0.1. Results Compared to CD19+CD20+ B cells, CD19+CD20- B cells: 1) were predominantly composed of DN (55% versus 19%, p &amp;lt; 0.01) and IgD-CD27+ memory subpopulations (37% versus 25%, p = 0.1094); 2) were detectable at a hierarchical frequency, RTX-T&amp;gt;RTX-N&amp;gt;HC cohorts; 3) had a greater frequency of Ki67+ cells (38% versus 5%, p &amp;lt; 0.01); 4) displayed higher CD38 MFI (41478.0 versus 10589.0, p &amp;lt; 0.01). Approximately 50% of CD19hi B cells were T-bet+, compared to 28% of CD19lo B cells (n = 33, p &amp;lt; 0.0001). Compared to CD19lo, CD19hi B cells 1) expressed (MFI) significantly greater TLR7, TLR9, IFNγR and pAMPK (n = 7 for all except pAMPK, n = 4), and 2) had greater frequency of IL6+ and IL10+ B cells (n = 7, p &amp;lt; 0.05) (Table). Conclusion CD20- B cell subpopulations with potent proliferative capacity express CD19. Further, CD19hi B cells: 1) upregulate the TLR-IFNγ-T-bet pathway; 2) have significantly greater expression of pAMPK; 3) have higher cytokine expression compared to CD19lo B cells. These data suggest the activation pathway, metabolic activity, and functional profile of CD19hi cells. Therefore, the potential of CD19hi cells to promote inflammation provides strong rationale for targeting CD19 in autoimmune disease with emerging therapies such as CAR-T cells and T cell engagers. Disclosure K. Shah: Grants/research support; KS has received funding for research from Roche. M.J. Leandro: Consultancies; MJL acted as a consultant for Roche and Genentech. Other; MJL received funds less than $10000 and support to attend educational conferences. M.S. Cragg: Consultancies; Consultant for BioInvent International, Boehringer Ingelheim, GSK, Radiant, iteos, Surrozen, Hanall and Mestag. Honoraria; He has received honoraria/speakers fees from Boehringer Ingelheim, Amgen, Roche and Symphogen. Grants/research support; BioInvent International, GSK and UCB. F. Kollert: Corporate appointments; FK is employed by Roche. Shareholder/stock ownership; FK has stock ownership at Roche. F. Schuler: Corporate appointments; FS is employed by Roche. Shareholder/stock ownership; FS has patents and stock ownership at Roche. D.A. Isenberg: None. A. Akbar: None. C. Klein: Corporate appointments; CK is employed by Roche. Shareholder/stock ownership; CK has patents and stock ownership at Roche. V. Reddy: Grants/research support; VR has received a research grant from Roche.

Multicomponent Pathogen-Mimicking Nanoparticles Induce Intestinal Immune Responses against Paratuberculosis.

Given the worldwide problem posed by enteric pathogens, the discovery of safe and efficient intestinal adjuvants combined with novel antigen delivery techniques is essential to the design of mucosal vaccines. In this work, we designed poly (lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) to codeliver all-trans retinoic acid (atRA), novel antigens, and CpG. To address the insolubility of the intestinal adjuvant atRA, we utilized PLGA to encapsulate atRA and form a "nanocapsid" with polydopamine. By leveraging polydopamine, we adsorbed the water-soluble antigens and the TLR9 agonist CpG onto the NPs' surface, resulting in the pathogen-mimicking PLPCa NPs. In this study, the novel fusion protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis antigens HBHA, Ag85B, and Bfra, was coloaded onto the NPs. In vitro, PLPCa NPs were shown to promote the activation and maturation of bone marrow-derived dendritic cells. Additionally, we found that PLPCa NPs created an immune-rich microenvironment at the injection site following intramuscular administration. From the results, the PLPCa NPs induced strong IgA levels in the gut in addition to enhancing powerful systemic immune responses. Consequently, significant declines in the bacterial burden and inflammatory score were noted in PLPCa NPs-treated mice. In summary, PLPCa can serve as a novel and safe vaccine delivery platform against gut pathogens, such as paratuberculosis, capable of activating both systemic and intestinal immunity.

TLR5 agonist in combination with anti-PD-1 treatment enhances anti-tumor effect through M1/M2 macrophage polarization shift and CD8+ T cell priming

Immune checkpoint inhibitors have revolutionized anti-tumor therapy, notably improving treatment responses in various tumors. However, many patients remain non-responsive and do not experience benefits. Given that Toll-like receptors (TLRs) can counteract tumor immune tolerance by stimulating both innate and adaptive immune responses, TLR agonists are being explored as potential immune adjuvants for cancer treatment. In this study, we assessed the potential of enhancing the efficacy of immune checkpoint inhibitors by activating innate immunity with a TLR5 agonist. In a mouse tumor model, combination therapy with TLR5 agonist and anti-PD-1 significantly inhibited tumor growth. The TLR5 agonist shifted the balance from M2-like to M1-like macrophages and upregulated the expression of co-stimulatory molecules in macrophages. Furthermore, TLR5 agonist promoted the activation and tumor infiltration of CD8+ T cells. As a result, the TLR5 agonist augmented the anti-tumor efficacy of anti-PD-1, suggesting its potential in modulating the tumor microenvironment to enhance the anti-tumor response. Our findings point toward the possibility of optimizing immune checkpoint inhibitor therapy using TLR5 agonists.

TLR Engagement Induces an Alternate Pathway for BCR Signaling that Results in PKCδ Phosphorylation.

Recently, we reported that preexposure of B cells to IL-4 induced an alternate, signalosome-independent BCR signaling pathway leading to protein kinase C (PKC)δ phosphorylation (pTyr311), which occurs in the membrane compartment. This is considered to represent a form of receptor crosstalk and signal integration. Unlike the classical BCR signaling pathway, Lyn kinase is indispensable for BCR-induced downstream events in the alternate pathway. Our previous report that alternate BCR signaling leading to ERK phosphorylation is triggered by LPS and PAM3CSK4 (much like IL-4) raises the possibility that other signaling outcomes such as PKCδ phosphorylation might be similarly affected. To explore the range of mediators capable of producing an alternate pathway for BCR signaling, we examined PKCδ translocation and phosphorylation in LPS- and PAM3CSK4-treated B cells stimulated by anti-Ig. We found that LPS and PAM3CSK4 alter the signaling pathway used by the BCR to produce PKCδ phosphorylation. As with IL-4, elements of the signalosome are not needed for PKCδ phosphorylation when BCR triggering occurs after LPS and PAM3CSK4. However, with LPS and PAM3CSK4, anti-Ig-induced phosphorylation of PKCδ takes place in the cytosol, in contrast to the IL-4-induced alternate pathway, wherein PKCδ phosphorylation occurs in the membrane. Furthermore, the BCR signaling pathway induced by LPS and PAM3CSK4 differs from that induced by IL-4 by not requiring Lyn. Thus, an alternate, signalosome-independent BCR signaling pathway for PKCδ phosphorylation is induced by TLR agonists but differs in important ways from the alternate pathway induced by IL-4.

Vibrational spectroscopy coupled with machine learning sheds light on the cellular effects induced by rationally designed TLR4 agonists

In this work, we present the potential of Fourier transform infrared (FTIR) microspectroscopy to compare on whole cells, in an unbiased and untargeted way, the capacity of bacterial lipopolysaccharide (LPS) and two rationally designed molecules (FP20 and FP20Rha) to activate molecular circuits of innate immunity. These compounds are important drug hits in the development of vaccine adjuvants and tumor immunotherapeutics. The biological assays indicated that FP20Rha was more potent than FP20 in inducing cytokine production in cells and in stimulating IgG antibody production post-vaccination in mice. Accordingly, the overall significant IR spectral changes induced by the treatment with LPS and FP20Rha were similar, lipids and glycans signals being the most diagnostic, while the effect of the less potent molecule FP20 on cells resulted to be closer to control untreated cells. We propose here the use of FTIR spectroscopy supported by artificial intelligence (AI) to achieve a more holistic understanding of the cell response to new drug candidates while screening them in cells.

Differential susceptibility of cells infected with defective and intact HIV proviruses to killing by obatoclax and other small molecules.

Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses. To investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals. We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay). Obatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects. Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

The E3 Ubiquitin Protein Ligase LINCR Amplifies the TLR-Mediated Signals through Direct Degradation of MKP1.

Toll-like receptors (TLRs) induce innate immune responses through activation of intracellular signaling pathways, such as MAP kinase and NF-κB signaling pathways, and play an important role in host defense against bacterial or viral infections. Meanwhile, excessive activation of TLR signaling leads to a variety of inflammatory disorders, including autoimmune diseases. TLR signaling is therefore strictly controlled to balance optimal immune response and inflammation. However, its balancing mechanisms are not fully understood. In this study, we identified the E3 ubiquitin ligase LINCR/ NEURL3 as a critical regulator of TLR signaling. In LINCR-deficient cells, the sustained activation of JNK and p38 MAPKs induced by the agonists for TLR3, TLR4, and TLR5, was clearly attenuated. Consistent with these observations, TLR-induced production of a series of inflammatory cytokines was significantly attenuated, suggesting that LINCR positively regulates innate immune responses by promoting the activation of JNK and p38. Interestingly, our further mechanistic study identified MAPK phosphatase-1 (MKP1), a negative regulator of MAP kinases, as a ubiquitination target of LINCR. Thus, our results demonstrate that TLRs fine-tune the activation of MAP kinase pathways by balancing LINCR (the positive regulator) and MKP1 (the negative regulator), which may contribute to the induction of optimal immune responses.

Abstract LT08: Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy

Abstract As our understanding of the complex interactions between the immune system and cancer has progressed, so has the identification of novel targets and ligands that can modulate effective anti-cancer immune responses. While immunotherapy has emerged as a mainstay in cancer therapy, its efficacy has been limited by the potential for off-target toxicity against normal body cells. In this study, we present a nanoparticle-based delivery system that enhances the signaling efficacy of immunomodulatory ligands while concurrently limiting systemic toxicity. We demonstrate that extracellular vesicles (EVs), lipid bilayer enclosed nanoparticles released by cells, can be efficiently engineered to display multiple immunomodulatory ligands on their surface. Display of immunomodulatory ligands such as agonistic CD137 and CD40 antibodies on the EV surface significantly enhanced their therapeutic efficacy over equivalent doses of free ligands by inducing ligand multimerization, efficient receptor crosslinking and the formation of immune synapses. EVs conjugated with a complementary combination of immune stimulatory ligands and immune checkpoint inhibitors were able to shift the tumor immune milieu towards an anti-tumorigenic phenotype in a lung metastatic melanoma allograft and suppress tumor progression to a greater extent than an equivalent dose of free ligands. We also demonstrate the ability of EVs to co-deliver intraluminally loaded TLR agonists which synergize with surface displayed agonistic CD40 antibodies to induce effective anti-tumor responses and tumor regression in a metastatic autochthonous cell-derived model of PDAC. Remarkably, we also observed that EV-associated immunotherapeutics were limited in their biodistribution to the tumor microenvironment, resulting in significantly lower off-target toxicity than treatment with free ligands. In summary, we present an EV-based delivery approach that allows the use of potent cancer immunotherapy combinations to achieve superior anti-tumor responses at significantly lower doses with less side-effects than is possible with conventional administration methods for cancer immunotherapy. Citation Format: Migara Kavishka Jayasinghe, Dawn Liu Xiao Tian, Yock Sin Lay, Chang Yu Lee, Brendon Zhi Jie Yeo, Dong Van Hoang, Rebecca Carissa Prajogo, Boya Peng, Thach Tuan Pham, Minh Thi Nguyet Le. Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT08.

Single-B cell analysis correlates high-lactate secretion with stress and increased apoptosis

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSRhigh B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSRhigh cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Size Tuning of Mesoporous Silica Adjuvant for One-Shot Vaccination with Long-Term Anti-Tumor Effect.

Despite recent clinical successes in cancer immunotherapy, it remains difficult to initiate a long-term anti-tumor effect. Therefore, repeated administrations of immune-activating agents are generally required in most cases. Herein, we propose an adjuvant particle size tuning strategy to initiate a long-term anti-tumor effect by one-shot vaccination. This strategy is based on the size-dependent immunostimulation mechanism of mesoporous silica particles. Hollow mesoporous silica (HMS) nanoparticles enhance the antigen uptake with dendritic cells around the immunization site in vivo. In contrast, hierarchically porous silica (HPS) microparticles prolong cancer antigen retention and release in vivo. The size tuning of the mesoporous silica adjuvant prepared by combining both nanoparticles and microparticles demonstrates the immunological properties of both components and has a long-term anti-tumor effect after one-shot vaccination. One-shot vaccination with HMS-HPS-ovalbumin (OVA)-Poly IC (PIC, a TLR3 agonist) increases CD4+ T cell, CD8+ T cell, and CD86+ cell populations in draining lymph nodes even 4 months after vaccination, as well as effector memory CD8+ T cell and tumor-specific tetramer+CD8+ T cell populations in splenocytes. The increases in the numbers of effector memory CD8+ T cells and tumor-specific tetramer+CD8+ T cells indicate that the one-shot vaccination with HMS-HPS-OVA-PIC achieved the longest survival time after a challenge with E.G7-OVA cells among all groups. The size tuning of the mesoporous silica adjuvant shows promise for one-shot vaccination that mimics multiple clinical vaccinations in future cancer immunoadjuvant development. This study may have important implications in the long-term vaccine design of one-shot vaccinations.

Toll-like receptors (TLR-2 and TLR-4): putative role in the uterine inflammatory response in cows – A mini review

Metritis and endometritis are the most prevalent uterine inflammatory diseases in bovines and cause infertility and high economic loss to farmers. Many studies have been conducted to investigate the mechanism of pathology, immune response and therapeutics associated with these diseases. This review focuses mainly on the immune system present in the uterus and on the involvement of Toll-like receptors. This study aimed to provide a deep understanding of the structure, function and expression pathways of Toll-like receptors (TLR-2 and TLR-4) in the treatment of pathogenic microorganisms invading the uterus and their significance in inflammatory conditions such as metritis and clinical and subclinical endometritis. The genetic associations of TLRs with diseases and some therapeutic interventions are also briefly discussed. As such, TLRs-2 and 4 play a critical role in providing a uterine defense mechanism against foreign substances (embryos, sperm, and pathogenic bacteria). In the present era, the use of TLR agonists is being unfurled for treatment, and further exploration and study of this topic in the future are needed.

Development of Non-toxic Recombinant Ricin Vaccine and Evaluation of Vaccine Efficacy

Ricin is a highly toxic protein which is produced in the seeds of the castor oil plant. Ricin toxin A chain has ribosomal RNA N-glycosylase activity that irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 within the 28S rRNA. In this study, we developed non-toxic recombinant ricin vaccine(R51) in &lt;i&gt;E. coli&lt;/i&gt; expression system, and evaluated efficacy of the R51 according to adjuvants. When the R51 was administered using aluminum hydroxide as an adjuvant, the vaccine efficacy was higher than that of TLR agonists or aluminum phosphate. Because it is time-consuming to administer the vaccine three times at three-week intervals, we investigated the survival rate and antibody titer of mice according to the change of time interval of vaccination. Interestingly, there was no difference in survival rate and antibody titer when R51 was administered at 0, 1, and 3 weeks or 0, 2, and 4 weeks compared to when administered at 0, 3, and 6 weeks. Therefore, the developed R51 vaccine is promising to protect soldiers from Ricin attack.