TLR Agonists Research Articles

Nano-Pulse Stimulation Treatment Inhibits Pan02 Murine Pancreatic Tumor Growth and Induces a Long-Term Adaptive Immune Response with Abscopal Effects When Combined with Immune-Enhancing Agents.

Pancreatic cancer is associated with a poor prognosis and immunotherapy alone has not demonstrated sufficient efficacy in the treatment of nonresectable tumors. Nano-Pulse Stimulation™ therapy (NPS™) applies nanosecond electric pulses that lead to regulated cell death, exposing tumor antigen to the immune system. To establish a primary Pan02 tumor, mice were intradermally injected with Pan02 cells into the right flank. Secondary, rechallenge tumors and distal, secondary tumors (abscopal response) were established by injecting Pan02 cells into the opposite left flank. After 5 days of tumor growth, one of the tumors was treated with NPS, followed by injection with an immune-enhancing agent to stimulate an immune response. Growth of the treated primary tumor and untreated rechallenge tumors (injected 60-days post-treatment) or distal secondary tumors (injected simultaneously with the primary) was monitored. NPS in combination with the adjuvant and TLR agonist, resiquimod (RES), was the optimal treatment regimen for both eliminating a primary Pan02 tumor as well as inhibiting growth of a Pan02 cell rechallenge tumor. This inhibition of the rechallenge tumor injected 2 months after eliminating the primary tumor suggests a long-term immune response had been stimulated. Additional support for this came from the observations that depleting CD8+ T-cells reduced inhibition of rechallenge tumor growth by 35% and rechallenge tumors had 3-fold more CD8+ T-cells than tumors injected after surgical resection of the primary tumor. When the NPS-treated tumor was immediately injected with the anti-OX40 antibody to agonize the function of the costimulatory T cell receptor, OX40, up to 80% of untreated abscopal tumors were eliminated. NPS plus RES was the most effective at both eliminating a primary tumor and inhibiting a rechallenge tumor. NPS treatment followed by injection of aOX40 was the most effective at inhibiting the growth of an untreated abscopal tumor.

TLR7 in channel catfish (Ictalurus punctatus) is expressed in the endolysosome and is stimulated by synthetic ssRNA analogs, imiquimod, and resiquimod.

Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms.

Human and Murine Toll-like Receptor-Driven Disease in Systemic Lupus Erythematosus.

The pathogenesis of systemic lupus erythematosus (SLE) is linked to the differential roles of toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. TLR7 overexpression or gene duplication, as seen with the Y-linked autoimmune accelerator (Yaa) locus or TLR7 agonist imiquimod, correlates with increased SLE severity, and specific TLR7 polymorphisms and gain-of-function variants are associated with enhanced SLE susceptibility and severity. In addition, the X-chromosome location of TLR7 and its escape from X-chromosome inactivation provide a genetic basis for female predominance in SLE. The absence of TLR8 and TLR9 have been shown to exacerbate the detrimental effects of TLR7, leading to upregulated TLR7 activity and increased disease severity in mouse models of SLE. The regulatory functions of TLR8 and TLR9 have been proposed to involve competition for the endosomal trafficking chaperone UNC93B1. However, recent evidence implies more direct, regulatory functions of TLR9 on TLR7 activity. The association between age-associated B cells (ABCs) and autoantibody production positions these cells as potential targets for treatment in SLE, but the lack of specific markers necessitates further research for precise therapeutic intervention. Therapeutically, targeting TLRs is a promising strategy for SLE treatment, with drugs like hydroxychloroquine already in clinical use.

Structure-Activity Relationship Analysis of Fluoxetine for Suppression of Inflammatory Cytokine Production.

There is accumulating evidence that selective serotonin reuptake inhibitors (SSRIs), clinically used as antidepressants, have a beneficial effect on inflammatory diseases such as coronavirus disease 2019 (COVID-19). We previously compared the inhibitory effects of five U.S. Food and Drug Administration (FDA)-approved SSRIs on the production of an inflammatory cytokine, interleukin-6 (IL-6), and concluded that fluoxetine (FLX) showed the most potent anti-inflammatory activity. Here, we investigated the structure-activity relationship of FLX for anti-inflammatory activity towards J774.1 murine macrophages. FLX suppressed IL-6 production induced by the TLR3 agonist polyinosinic-polycytidylic acid (poly(I : C)) with an IC50 of 4.76 µM. A derivative of FLX containing chlorine instead of the methylamino group lacked activity, suggesting that the methylamino group is important for the anti-inflammatory activity. FLX derivatives bearing an N-propyl or N-(pyridin-3-yl)methyl group in place of the N-methyl group exhibited almost the same activity as FLX. Other derivatives showed weaker activity, and the N-phenyl and N-(4-trifluoromethyl)benzyl derivatives were inactive. The chlorine-containing derivative also lacked inhibitory activity against TLR9- or TLR4-mediated IL-6 production. These derivatives showed similar structure-activity relationships for TLR3- and TLR9-mediated inflammatory responses. However, the activities of all amino group-containing derivatives against the TLR4-mediated inflammatory response were equal to or higher than the activity of FLX. These results indicate that the substituent at the nitrogen atom in FLX strongly influences the anti-inflammatory effect.

An LGR6 frameshift variant abrogates receptor expression on select leukocyte subsets and is associated with viral infections

AbstractThe leucine-rich repeat-containing G-protein–coupled receptor 6 (LGR6) was recently identified as the cognate receptor for the proresolving mediator maresin 1 (MaR1). To address the biological role of LGR6 in humans, we investigated the functional impact of a genetic variant in the gene encoding for LGR6, which is predicted to lead to a frameshift mutation in one of the receptor isoforms, on both receptor expression and immune cell responses. In neutrophils, monocytes, and natural killer (NK) cells from volunteers homozygous for this variant, we found a significant downregulation in the expression of LGR6 when compared with controls without the variant; whereas the LGR6 expression was essentially similar in monocyte-derived macrophages and CD8+ T cells. Functionally, loss of LGR6 expression was linked with a decreased ability of neutrophils and monocytes to phagocytose bacteria. We observed an increase in neutrophil chemotaxis and leukotriene B4 production and increased expression of activation markers, including markers for platelet-leukocyte phagocyte heterotypic aggregates, such as CD41, in neutrophils and monocytes from the variant group. Using data from the UK Biobank, we found that at a population level the rs4266947 variant, which is in high linkage disequilibrium with rs74355478, was associated with a higher incidence of viral infections. Intriguingly, neutrophils, NK cells, and CD8+ T cells from volunteers with the LGR6 variant displayed altered viral responses when stimulated with Toll-like receptor 3 (TLR3), TLR7/TLR8, and TLR9 agonists. Together, these findings shed new light on the cell type–specific regulation of LGR6 expression and the role of this receptor in directing host immune responses.

Selective production of IL-33-neutralizing autoantibody ameliorates asthma responses and severity

BackgroundNatural anti-cytokine autoantibodies can regulate homeostasis of infectious and inflammatory diseases. The anti-cytokine autoantibody profile and relevance to the pathogenesis of asthma are unknown. We aim to identify key anti-cytokine autoantibodies in asthma patients, and reveal their immunological function and clinical significance. MethodsA Luciferase Immunoprecipitation System was used to screen serum autoantibodies against 11 key cytokines in patients with allergic asthma and healthy donors. The antigen-specificity, immunomodulatory functions and clinical significance of anti-cytokine autoantibodies were determined by ELISA, qPCR, neutralization assays and statistical analysis, respectively. Potential conditions for autoantibody induction were revealed by in vitro immunization. ResultsOf 11 cytokines tested, only anti-IL-33 autoantibody was significantly increased in asthma, compare to healthy controls, and the proportion positive was higher in patients with mild-to-moderate than severe allergic asthma. In allergic asthma patients, the anti-IL-33 autoantibody level correlated negatively with serum concentration of pathogenic cytokines (e.g., IL-4, IL-13, IL-25 and IL-33), IgE, and blood eosinophil count, but positively with mid-expiratory flow FEF25–75%. The autoantibodies were predominantly IgG isotype, polyclonal and could neutralize IL-33-induced pathogenic responses in vitro and in vivo. The induction of the anti-IL-33 autoantibody in blood B-cells in vitro required peptide IL-33 antigen along with a stimulation cocktail of TLR9 agonist and cytokines IL-2, IL-4 or IL-21. ConclusionsSerum natural anti-IL-33 autoantibodies are selectively induced in some asthma patients. They ameliorate key asthma inflammatory responses, and may improve lung function of allergic asthma.

HIV rebound in HIV controllers is associated with a specific fecal microbiome profile.

HIV infection is associated with gut dysbiosis, and microbiome variability may affect HIV control when antiretroviral therapy (ART) is stopped. The TLR7 agonist, vesatolimod, was previously associated with a modest delay in viral rebound following analytical treatment interruption in HIV controllers (HCs). Using a retrospective analysis of fecal samples from HCs treated with vesatolimod or placebo (NCT03060447), people with chronic HIV (CH; NCT02858401) or without HIV (PWOH), we examined fecal microbiome profile in HCs before/after treatment, and in CH and PWOH. Microbiome diversity and abundance were compared between groups to investigate the association between specific phyla/species, immune biomarkers, and viral outcomes during treatment interruption. Although there were no significant differences in gut microbiome diversity between people with and without HIV, HCs, and CH shared common features that distinguished them from PWOH. there was a trend toward greater microbiome diversity among HCs. Treatment with vesatolimod reduced dysbiosis in HCs. Firmicutes positively correlated with T-cell activation, while Bacteroidetes and Euryarchaeota inversely correlated with TLR7-mediated immune activation. Specific types of fecal microbiome abundance (e.g. Alistipes putredinis) positively correlated with HIV rebound. In conclusion, variability in the composition of the fecal microbiome is associated with markers of immune activation following vesatolimod treatment and ART interruption.

TLR agonists polarize interferon responses in conjunction with dendritic cell vaccination in malignant glioma: a randomized phase II Trial

In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+ T-cells, while CD38 and CD39 expression are reduced on CD8+ T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+ T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Exploring malaria parasite surface proteins to devise highly immunogenic multi-epitope subunit vaccine for Plasmodium falciparum

BackgroundMalaria has remained a major health concern for decades among people living in tropical and sub-tropical countries. Plasmodium falciparum is one of the critical species that cause severe malaria and is responsible for major mortality. Moreover, the parasite has generated resistance against all WHO recommended drugs and therapies. Therefore, there is an urgent need for preventive measures in the form of reliable vaccines to achieve the target of a malaria-free world. Surface proteins are the preferable choice for subunit vaccine development because they are rapidly detected and engaged by host immune cells and vaccination-induced antibodies. Additionally, abundant surface or membrane proteins may contribute to the opsonization of pathogens by vaccine-induced antibodies. ResultsIn our study, we have listed all those surface proteins from the literature that could be functionally important and essential for infection and immune evasion of the malaria parasite. Eight Plasmodium surface and membrane proteins from the pre-erythrocyte and erythrocyte stages were shortlisted. Thirty-seven epitopes (B-cell, CTL, and HTL epitopes) from these proteins were predicted using immune-informatic tools and joined with suitable peptide linkers to design a vaccine construct. A TLR-4 agonist peptide adjuvant was added at the N-terminus of the multi-epitope series, followed by the PADRE sequence and EAAAK linker. The TLR-4 receptor was docked with the construct’s anticipated model structure. The complex of vaccine and TLR-4, with the lowest energy −1514, was found to be stable under simulated physiological settings. ConclusionThis study has provided a novel multi-epitope construct that may be exploited further for the development of an efficient vaccine for malaria.

Influence of TLR4 signaling on Cannabidiol’s antitumor effectiveness in lung adenocarcinoma cells

Abstract Objectives Lung cancer remains a predominant cancer type with high incidence and low survival rates. Key challenges in its treatment include impaired cellular mechanisms, notably resistance to apoptosis and altered immune responses. A critical aspect in this context is the heightened TLR4-mediated signaling, known to promote cell survival, metastasis, and resistance to cell death, particularly impacting immune microenvironment regulation. This study focuses on evaluating the impact of TLR4 signaling activation on potential therapeutic strategies. Methods Our research utilizes Cannabidiol (CBD), a compound already employed in mitigating chemotherapy side effects in lung adenocarcinoma, recognized for its antitumor properties including antiproliferative, antimetastatic, and apoptosis-inducing effects. However, the effectiveness of CBD in lung cancer cells with elevated TLR4 signaling remains uncertain. Results Our findings reveal that the combination of CBD and TLR4 agonist affects cell viability, proliferation, cell cycle progression, apoptosis, and gene expression related to immune response and extracellular matrix regulation. In lung adenocarcinoma cells with activated TLR4, CBD shows an increased IC50 value, reflecting reduced antiproliferative capacity. Furthermore, its efficacy in arresting the cell cycle and inducing apoptosis is also compromised. The influence on immune response and extracellular matrix regulation is also altered in TLR4-activated cells. Conclusions These results indicate that TLR4 activation significantly diminishes the antitumor efficacy of CBD. This highlights the importance of considering TLR4 signaling activation in future research on therapeutic agents like CBD for cancer treatment.

Abstract PO1-18-03: Chemokine-modulating regimen (rintatolimod, IFN-α2b, celecoxib): New strategy to drive CD8+T-cells into triple negative breast cancer

Abstract BACKGROUND: Pathologic complete response (pCR) is critical for positive long-term outcomes after neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) but is achieved only in 40-50% of patients. Combination of NAC with pembrolizumab, the new standard of care in TNBC, increases the pCR rate to 65% but is associated with significant and often permanent immune-related toxicities. Higher levels of CD8+ cytotoxic T-lymphocytes (CTLs) and low levels of regulatory T-cells (Treg) and myeloid derived suppressor cells (MDSC) in the tumor microenvironment (TME) predict improved relapse-free survival (RFS), overall survival (OS) and pCR, a surrogate marker for RFS. Locally produced chemokines CCL5, CXCL9, CXCL10 and CXCL11 are critical for CTL infiltration, while CCL22 is responsible for Treg attraction, with high CXCL9 being associated with a 3-fold higher rate of pCR after NAC. Our preclinical data show that Chemokine-modulatory (CKM) regimen, combining rintatolimod (TLR3 agonist), interferon (IFN)α2b and celecoxib (COX-2 inhibitor), eliminates the suppressive aspects of paclitaxel-induced inflammation and induces CTL-attractants but decreases MDSC- and Treg-attractants in the TME. We hypothesized that the combination of CKM with paclitaxel-based NAC will promote selective CTL infiltration into TNBC, and along with doxorubicin/cyclophosphamide (AC), will result in higher rate of pCR, translating into improved RFS and OS. METHODS: In this phase I study NCT04081389, 9 pts with stage I-III TNBC and median age of 47 (37-55) years received 3 weeks of paclitaxel with CKM, followed by 9 weeks of paclitaxel alone, followed by standard dose-dense AC and surgery. Paclitaxel was given weekly IV at 80 mg/m2 for 12 weeks, rintatolimod 200 mg IV, IFN-α2b and celecoxib 200 mg oral twice daily on days 1-3 of each of the initial 3 weeks. IFN-α2b was administered in an accelerated titration design at doses 0 or 5 million units (MU)/m2 [Dose Level (DL) 1 and 2, respectively] in the first 2 pts (no intra-patient dose escalation), then 10 MU/m2 [DL3] in 4 pts and then 20 MU/m2 [DL4] in 3 pts. Day 1 pre- and post-treatment blood samples were drawn. Pre- and post-treatment (at 3 weeks) biopsies were obtained from 5 pts at DL3 and DL4. Dose-limiting toxicity (DLT) was defined as grade 3 or higher within the first 3 weeks of treatment. Primary endpoint was safety and tolerability, to determine the recommended phase II dose (RP2D) of CKM for extended efficacy study. Secondary endpoints included efficacy (pCR), along with RFS and OS. Biomarkers were analyzed in exploratory studies. RESULTS: Treatment was well-tolerated with mostly grade 1 or 2 treatment-related adverse events (TRAEs) and no DLTs. Grade 3 TRAE were neutropenia (3/9), attributed to CKM (1/9) or paclitaxel (3/9), pneumonia (1/9) and anemia (1/9) attributed to AC. Paclitaxel- or AC-related toxicities were not higher than expected. There were no delayed or immune-related toxicities beyond treatment. 5/9 (56%) pts attained pCR, and 1 additional pt had ypTmic at the time of surgery. There was consistent (p=0.07) selective increase in CD8α (CTL marker) in post-treatment tumor biopsies with concomitant decrease in CD8α in the blood (p=0.04) measured by RT-PCR. RNA sequencing on the blood samples (7 pre- and post-treatment) showed that higher M2 macrophages and naïve B cells in the pre-treatment samples were associated with higher probability of non pCR (adjusted p=0.02 and 0.05, respectively). CKM treatment decreased CD8α and CD8β post-treatment in the blood samples. Higher CD8+ T cells and NK cells in the post-treatment blood samples, were associated with higher likelihood of pCR, although this trend did not reach statistical significance. CONCLUSIONS: We have observed preliminary indications of safety and good tolerability of the combination of CKM regimen with paclitaxel, with promising pCR + ypTmic of 66%. RNA sequencing indicates that CKM may drive CD8+ T cells from the blood into the tumor. Citation Format: Shipra Gandhi, Cayla Jones, Mateusz Opyrchal, Ronald Slomba, Kristopher Attwood, Jianmin Wang, Eduardo Cortes Gomez, Tracey O'Connor, Ellis Levine, Pawel Kalinski. Chemokine-modulating regimen (rintatolimod, IFN-α2b, celecoxib): New strategy to drive CD8+T-cells into triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-03.

Abstract PO2-27-04: Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer

Abstract Fibroblast Growth Factor Receptor 1 (FGFR1) has emerged as a critical player in various cancer types, including breast cancer. FGFR1 alterations, including gene amplification, overexpression, and activating mutations, have been identified in subsets of breast cancers, contributing to aberrant signaling pathways that promote tumor growth, angiogenesis, and metastasis. Our current research endeavors are directed towards unravelling the intricate mechanistic underpinnings of FGFR1's dual impact on breast tumor progression, specifically its contribution to endocrine resistance and its regulatory role in innate immune sensing and subsequent induction of Type 1 interferons (IFNs) Our investigative efforts have previously established a direct association between FGFR1 and endocrine resistance in hormone receptor positive T47D breast cancer cells. Moreover, a remarkable and distinct inverse correlation linking FGFR1 expression to the Type 1 IFN response was observed. We noticed that in vitro stimulation of T47D cells with Type 1 interferons induces the expression of FGFR1 and ESR1, thereby suppressing the interferon-stimulated genes. Interestingly, we observed that the FGFR1-mediated suppression of the Type 1 interferon response is associated with intracellular nucleic acid sensors, specifically toll-like receptors TLR7 and TLR9. Furthermore, we found that the inhibition of FGFR kinase enhances the expression of IFN-alpha, IFN-beta, TLR7, and TLR9 in T47D cells when stimulated with the TLR7 agonist loxoribine. These results strongly suggest that FGFR1 functions as an innate checkpoint and may be exploited by tumor cells to dampen Type 1 interferon responses, thereby inhibiting antitumor immunity in hormone receptor-positive breast cancer. Citation Format: Sushil Dhakal, Torbjørn Amundsen Lien, Jens Henrik Norum, Helga Bergholtz, Helene Midtun Flatekvål, Therese Sørlie. Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-04.

Abstract PS07-06: A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention

Abstract Purpose: To evaluate the merits of a novel citrullinated-enolase 1 (ENO1) peptide-based vaccine for inducing an effective response against triple negative breast cancer (TNBC). Background: Targets for cancer vaccines have included mutated proteins and peptides, as well as proteins that are overexpressed in cancer but otherwise ubiquitous in their structure. However, protein modifications other than mutation may also induce an immune response. Several lines of evidence have demonstrated a role for the posttranslational modification (PTM) citrullination as inducing immunogenicity. Dysregulated protein citrullination by peptidyl arginine deiminases (PADI) has been associated with autoimmune diseases and is currently being explored for its relevance to cancer. Previously, our group established PADI2, which catalyzes the PTM from arginine to citrulline, to be highly expressed in TNBC compared to normal mammary and other tissues. We observed that PADI2-associated protein citrullination promotes antigenicity. Importantly, we demonstrated that protein citrullination, non-occurring in DNA and RNA-based vaccines, is highly cancer specific. On the basis of these findings, we developed a novel vaccine based on cancer-selective citrullinated-enolase 1 peptides (cit-ENO1) that were identified from mass spectrometry surfaceome and immunopeptidome data from breast cancer cells. We assessed the extent to which a citENO1 peptide based vaccine elicits a potent host response and attenuates tumor development in a syngeneic mouse model of TNBC. Design: To confirm that the cit-ENO1 peptide vaccine elicited an immune response, we immunized female B6129SF1/J mice with 10nmol cit-ENO1 peptides or 10nmol of corresponding unmodified peptides as the antigen, plus the TLR3 agonist poly I:C as an adjuvant (10mg), subcutaneously once a week, for three weeks. Saline or the adjuvant alone were used as controls. One week post the last round of immunization, draining lymph nodes (LNs) and immunized skin were harvested, and T cell phenotypes evaluated using flow-cytometry. To assess anti-cancer efficacy, female B6129SF1/J mice were immunized with cit-ENO1 peptides or unmodified peptides with poly I:C as an adjuvant followed by orthotopic implantation with murine BRCA1co/co; MMTV-Cre; p53+/- TNBC cells (1.5 x 106) and tumor growth as well as overall survival were assessed. Results: We observed that the cit-ENO1 peptide vaccination induced a statistically significantly greater percentage of activated CD8+ PD-1+ effector T cells as well as CD8+ CD44+ CD62L- effector memory T cells in the LNs compared with either the control groups or the unmodified ENO1 peptide. CD8+ PD-1+ T-cells were also found to be significantly elevated in immunized skin of cit-ENO1 vaccinated mice. Both cit-ENO1 and unmodified ENO1 vaccine increased CD4+ PD-1+ T cells from the LNs and immunized skin. Remarkably, the cit-ENO1 vaccine but not the unmodified-ENO1 vaccine significantly delayed tumor growth and markedly improved overall survival compared to respective controls (Logrank test for trend 2-sided p< 0.05). Conclusion: We have developed a novel vaccine based on cancer-selective cit-ENO1 peptides that stimulates a potent anti-cancer immune response and significantly delays tumor initiation and progression in a preclinical, immune competent mouse model of TNBC. Importantly, the anti-cancer effects were not observed with corresponding unmodified ENO1 peptides. Citation Format: Ricardo Leon Letelier, Hiroyuki Katayama, Yihui Chen, Yinning Cai, Fuchung Hsiao, Banu Arun, Samir Hanash. A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-06.

A combination of TLR9 and STING agonists favors antigen cross-presentation and promotes Th1 immune response against influenza virus

Abstract Adjuvants can boost the type, magnitude, breadth, and durability of vaccine responses. We have previously demonstrated that combining adjuvants through rational design can overcome age-specific impairments in inducing Th1 immunity, trigger antigen cross-presentation, and enable CD8-driven protective immunity of protein-based vaccine formulations. Here, we screened 78 adjuvant combinations to explore their molecular mechanism of action through human in vitro modeling and identified combinations that synergistically promote the expansion of antigen-specific CD4+ cells, induce cross-presentation on MHC class I resulting in activation of antigen-specific CD8+ cells, and redirect the type of immune response to favor the production of Th1-promoting cytokines. The combination of CpG (TLR9 agonist) and 2,3-cGAMP (STING agonist) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored the secretion of Th1-polarizing cytokines TNF and IL-12p70. Activation of T-bet-expressing Th1 cells was also observed with this adjuvant combination. We also demonstrate the phenotypic reprogramming towards conventional type 1 dendritic cells by CpG + 2,3-cGAMP, and the ability of this adjuvant combination to induce similar immune responses in immune cells from newborns and older adults. Identification of the adjuvant combination CpG + 2,3-cGAMP may prove key to the future development of vaccines against intracellular pathogens such as respiratory viruses.

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.