Induction Of Immune Responses Research Articles

TLR-mediated immune activation is a strong suppressor of the Treg-attracting chemokine CCL22

Abstract The immunoregulatory chemokine CCL22 is mainly secreted by dendritic cells (DC) in lymph nodes and plays a crucial role in maintaining the balance between immune tolerance and efficient immune response by induction of intercellular contacts between DC and regulatory T cells (Treg) expressing the only so far known CCL22 receptor CCR4. In the absence of CCL22, the immune response to vaccination and cancer has been proven to be enhanced. In this work, we aimed to understand how activation of Toll-like receptors (TLR) modulates CCL22 levels mechanistically in vitro and in vivo. We observed that a bacterial infection with salmonella typhimurium and treatment with different TLR agonists such as Poly (I:C), LPS, R848 and CpG led to a potent and long-lasting suppression of systemic CCL22 in vivo. Furthermore, using in vitro co-cultures of DC, B cells and T cells, we identified TLR-mediated CCL22 suppression to be mediated by soluble factors derived from non-DC. Additionally, we pursued to investigate the underlying mechanism and could show in vitro that CCL22 suppression is particularly driven by redundant action of IL-10, IFN-α and IFN-γ. Moreover, inhibition of NF-κB reverted the CCL22-suppressing effect of CpG on splenocytes. In CpG-treated mice, Treg/DC contacts were diminished to the level known from Ccl22 −/−mice, an observation which links TLR-mediated CCL22 suppression to an immunological relevance. Taken together, we demonstrate that TLR signaling results in CCL22 downregulation via different cytokines, a mechanism which is potentially important to enable the induction of a strong immune response and efficient pathogen clearance in the setting of microbial infection.

3D co-culture models demonstrate radiation-mediated activation of tumor-associated macrophages

Abstract Type I IFN signaling mediated by STING pathway is important for the induction of radiation-stimulated adaptive immune responses in responsive tumors. Macrophages recruited to tumors following radiotherapy (RT) can influence the response to RT. Reprogramming immunosuppressive M2-like macrophages in tumors to exhibit a more activated M1-like phenotype can help improve response to therapies. Here, we hypothesize that a 3D co-culture model of cancer cells and macrophages will be more contextually relevant to the events occurring in tumors and can be used to study the crosstalk that occurs between these cells in vivo. We initially optimized seeding densities of murine MC38 colorectal carcinoma cells to develop spheroids of consistent size. Using GFP+ macrophages and Hoechst stained MC38, we imaged the development and localization of these cells during spheroid formation. Next, co-culture spheroids were exposed to 12 Gy radiation on day 1. Viability and phenotype of macrophages in spheroid co-cultures was assessed on day 4 using flow cytometry by staining for CD80, CD86, MHC Class II, CD69 and CD206 expression. STING ligand, cyclic di-AMP (CDA) treated spheroid co-cultures were used as positive controls. CDA treatment significantly increased CD80/86 expression in 3D co-cultures. Treatment with 12 Gy radiation showed similar effect. Interestingly, macrophages derived from MyD88 Lyz2Cre, MyD88 fl/fland STING knockout mice also showed a significant increase in CD80/86 and CD40, suggesting that macrophage activation was independent of MyD88 or STING signaling. Ongoing studies aim to explore the pathways activated on irradiation in these cells and further cross-validate with the macrophage phenotypes observed in vivo in response to RT.

Monoclonal anti-toxin therapy supports the protective innate immune response following Clostridioides difficileinfection

Abstract Clostridioides difficile causes over 200,000 hospitalizations and 13,000 deaths annually in the United States. C. difficile infects the large intestine following perturbation of the microbiome to cause pathology that ranges in severity from diarrhea to pseudomembranous colitis. Virulence factors Toxin A and Toxin B are the primarily drivers of disease. These toxins damage the epithelial barrier leading to pathologic inflammation and bacterial translocation. Monoclonal antibodies (mAbs) that neutralize Toxin A and Toxin B, actoxumab and bezlotoxumab, respectively, significantly reduce disease severity in a murine model of C. difficile infection, however the impact of toxin neutralization on the induction of the innate immune response following infection is unknown. The goal of this study was to define the quality of the host innate immune response in the context of anti-toxin mAb therapy. At day 2 post-infection, C. difficile infected, mAb-treated mice had significantly less disease despite no differences in C. difficile burden compared to isotype-treated mice. Further, no difference in neutrophil infiltration or production of IFNg or IL-17 from innate lymphoid cells (ILCs) was observed within the intestinal lamina propria following infection. However, C. difficile-infected, mAb-treated mice had increased IL-22-producing ILCs and increased eosinophil infiltration in the intestine compared to iso-treated mice. Both IL-22 +ILCs and eosinophils promote protective host response following C. difficile infection. These findings indicate that activation of natural host protective mechanisms remain intact in the context of treatment that neutralizes toxin but does not alter C. difficile burden in the intestine. Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA

Intracellular Monitoring by Dendritic Cells – a New Way to Stay Informed – From Simple Scavenger to Active Gatherer.

Abstract The ability of dendritic cells (DCs) to acquire antigen from their environment is critical for the induction of an adaptive immune response and the maintenance of tolerance. Despite its importance, it is tacitly assumed that DCs acquire antigen by scavenging extracellular material via endocytosis or macropinocytosis. Here, using labeled RNA and time lapse imaging, we observe DCs siphoning material directly from live neighboring keratinocytes. This contact dependent mechanism of antigen uptake – which we term intracellular monitoring– accounts for a large percentage of RNA transferred to DCs, and cannot be blocked by inhibitors of known antigen uptake processes. It is exclusive to DCs, as other cell types do not efficiently siphon RNA from neighboring cells, and common among most DCs subsets tested. Langerhans cells with a conditional gene deletion can successfully overcome their deficiency by siphoning RNA and protein from their neighbors, but only if the deleted protein is expressed in the donating cell. Such behavior reframes our understanding of DCs from mere scavengers to highly informed supervisors with extensive access to the cytosol of surrounding cells. Intracellular monitoring may therefore play important roles in the maintenance of tolerance, defense against infection, and detection of cancerous cells.

Lymphoid Follicle Transcriptome Reveals Maladaptive Immune Responses In Severe Chronic Obstructive Pulmonary Disease (COPD).

Abstract Rationale: Increases in the number and size of lymphoid follicles (LFs) are present in severe stages of COPD and have not been fully characterized. HLA complex is crucial in enabling the immune system to recognize “self” versus “non-self” antigens and plays a major role in immune responses to cigarette smoke exposure. Methods: Lung follicles from COPD patients with milder (GOLD I–II) and more severe (GOLD III–IV) COPD were sampled using GeoMX® Digital Spatial Transcriptional Profiling. Data were analyzed and normalized using GeoMx® DSP Analysis Suite. Results: Pathway analysis showed that B cell activation pathways had the highest fold enrichment. GOLD I–II compared to GOLD III–IV COPD patients had the highest expression of HLA-Cand its ligands (KIR2DS2, KIR2DS1, KIR3DL1; p<0.0001) which facilitate CD8-mediated cytotoxicity, and HLA-Gand its receptor LILRB4(p=0.01) which are key modulators of immune tolerance. GOLD III–IV COPD patients had the highest HLA enrichment in LFs, with upregulation of transcription factors (HIF1A, IRF1, NFYBand NFYC) and assembly components (CALR, TAPBP, ERP27, MARCHF1, B2M, and CIITA) of HLA (p<0.01). Compared to GOLD I–II, LFs from GOLD III–IV COPD patients had much higher expression of HLAs involved in autoimmune responses to self-antigens and adaptive immune evasion, such HLA-DRA, HLA-DRB1, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-Eand HLA-B(p<0.0001). Conclusions: We show for the first time that LFs from patients with severe COPD are characterized by expression of HLAs associated with immune evasion and autoimmune responses, whereas LFs in milder stages of COPD are characterized by induction of immune responses controlled by tolerance mechanisms R01 HL 149744-04/06

The Role of TSLP in Atopic Dermatitis: From Pathogenetic Molecule to Therapeutical Target.

Atopic dermatitis (AD) is a kind of chronic skin disease with inflammatory infiltration, characterized by skin barrier dysfunction, immune response dysregulation, and skin dysbiosis. Thymic stromal lymphopoietin (TSLP) acts as a regulator of immune response, positively associated with AD deterioration. Mainly secreted by keratinocytes, TSLP interacts with multiple immune cells (including dendritic cells, T cells, and mast cells), following induction of Th2-oriented immune response during the pathogenesis of AD. This article primarily focuses on the TSLP biological function, the relationship between TSLP and different cell populations, and the AD treatments targeting TSLP.

High larval densities and high temperatures lead to a stronger immune response in the black soldier fly

Organisms are expected to invest more in their immune function when the risk of disease infection is high. However, induction of a robust immune response is costly and may not be achievable in suboptimal environments. High conspecific density could simultaneously imply high infection risk and a suboptimal environment for many insect species. We focus on the economically important dipteran species (black soldier fly, BSF) that represents the insect order that has been ignored in previous research on density effects on immunity. The experimental part of the study was carried out to evaluate the effect of larval density (three density treatments: 1, 5 and 10 larvae/cm2) and temperature (three thermal treatments: 23, 27 and 30 °C) on the immune function of BSF larvae. The larvae that were reared at high compared to low larval densities and at higher than lower temperatures had significantly higher activity of phenoloxidase, an enzyme that plays an essential role in insect immune function. Sex did not have a significant effect on phenoloxidase activity and prepupal mass, pupal mass and adult mass were not affected by the levels of phenoloxidase activity of fifth instar larvae. In addition, we give an overview of larval density effects on insect immunity and show that density-dependent prophylaxis (stronger immune response in high larval density environments) is indeed common in the results of published case studies. However, cases with no correlation between density and immunity traits were as frequent. Moreover, in more than half of the studies, qualitatively different within-species patterns in different immunity traits were observed. We conclude that BSF larvae exhibit density-dependent prophylaxis, and larvae invest more into their immune system at high larval densities and temperatures than they do at low larval densities and temperatures.

A genome-wide optical pooled screen reveals regulators of cellular antiviral responses

The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with insitu cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells-over an order of magnitude more genomic perturbations than demonstrated previously using an insitu SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches.

Exposure of adult zebrafish (Danio rerio) to SARS-CoV-2 at predicted environmentally relevant concentrations: Outspreading warns about ecotoxicological risks to freshwater fish

While the multifaceted social, economic, and public health impacts associated with the COVID-19 pandemic are known, little is known about its effects on non-target aquatic ecosystems and organisms. Thus, we aimed to evaluate the potential ecotoxicity of SARS-CoV-2 lysate protein (SARS.CoV2/SP02.2020.HIAE.Br) in adult zebrafish (Danio rerio) at predicted environmentally relevant concentrations (0.742 and 2.226 pg/L), by 30 days. Although our data did not show locomotor alterations or anxiety-like or/and anxiolytic-like behavior, we noticed that exposure to SARS-CoV-2 negatively affected habituation memory and social aggregation of animals in response to a potential aquatic predator (Geophagus brasiliensis). An increased frequency of erythrocyte nuclear abnormalities was also observed in animals exposed to SARS-CoV-2. Furthermore, our data suggest that such changes were associated with a redox imbalance [↑ROS (reactive oxygen species), ↑H2O2 (hydrogen peroxide), ↓SOD (superoxide dismutase), and ↓CAT (catalase)], cholinesterasic effect [↑AChE (acetylcholinesterase) activity], as well as the induction of an inflammatory immune response [↑NO (nitric oxide), ↑IFN-γ (interferon-gamma), and ↓IL-10 (interleukin-10)]. For some biomarkers, we noticed that the response of the animals to the treatments was not concentration-dependent. However, principal component analysis (PCA) and the “Integrated Biomarker Response” index (IBRv2) indicated a more prominent ecotoxicity of SARS-CoV-2 at 2.226 pg/L. Therefore, our study advances knowledge about the ecotoxicological potential of SARS-CoV-2 and reinforces the presumption that the COVID-19 pandemic has negative implications beyond its economic, social, and public health impacts.

Abstract 689: The codon-modified influenza virus CodaLytic™rescues immunostimulatory activity and anti-tumor efficacy in PD-1-refractory tumor models

Abstract Multiple oncolytic viruses have been shown to induce various beneficial changes in the tumor microenvironment (TME), which ultimately support induction of anti-tumor immune responses. We have previously demonstrated that CodaLytic, a codon-modified influenza virus, can kickstart the cancer immunity cycle at various steps in several mouse models with differing baseline immune contextures. Here, we are confirming the mechanisms of action of the virus in combination with immune checkpoint inhibition in murine and human preclinical models. Efficacy after intratumoral injection of 108 PFU CodaLytic in combination with αPD-1 and/or αCTLA-4 checkpoint blockade was determined in αPD-1-resistant B16-F10 melanoma and 4T1 triple-negative breast cancer mouse models. Changes in the tumor immune infiltrate after treatment were characterized using flow cytometry. Primary human tumoroids were incubated with CodaLytic +/- 10 μg/ml pembrolizumab using the 3D-Explore platform by Nilogen Oncosystems. Tumor cell killing (TCK) was detected by high-content imaging at 72h and effects on the TME were characterized by cytokine release and RNA sequencing at 24h and/or 48h. In B16-F10, addition of CodaLytic to αPD-1 blockade rescued non-significant tumor growth inhibition (TGI) with αPD-1 alone, achieving 86% TGI (p < 0.01), 40% complete regressions and 60% survival beyond 45 days (69% TGI, 20% regressions and 30% long-term survival after CodaLytic monotherapy). Mechanistically, CodaLytic-containing regimens increased tumor immune infiltration, in particular with CD8+ T cells and cross-presenting dendritic cells (DCs). 4T1 tumors did not significantly respond to αPD-1, αCTLA-4 or CodaLytic monotherapies, but achieved 86% TGI in combination with 50% long-term survival past 55 days. CTLA-4 blockade emerged as the initial driver of slowed tumor growth and αPD-1 drove long-term survival, which was further augmented by CodaLytic addition. Infiltration with CD45+ leukocytes and cross-presenting DCs correlated with tumor volumes. The benefit of CodaLytic combination treatment was also demonstrated ex vivo in human tumoroid cultures. Meaningful TCK was observed in 50% of specimens vs 17% with pembrolizumab and 33% with CodaLytic alone. In this system without immune cell recruitment from a systemic reservoir, sustained release of CXCL9 and CXCL10 and temporal decreases of MIP-1β and CCL2 associated with response. Additional transcriptional analyses are ongoing. Taken together, this and additional preclinical data confirms the emergence of CodaLytic as a potent immunostimulatory agent capable of restoring immune cell infiltration and anti-tumor efficacy when used in combination with checkpoint inhibition in PD-1 resistant models. This data supports future development of CodaLytic for immuno-virotherapy of tumor types in which checkpoint therapy is less established. Citation Format: Yiwen Zhao, Nusrat Jahan, Marcin Stawowczyk, Katie Pfeffer, Juliana Tafrova, James Rodriguez, Chen Yang, Sybil A. Tasker, Steffen Mueller, J. Robert Coleman, Johanna K. Kaufmann. The codon-modified influenza virus CodaLytic™rescues immunostimulatory activity and anti-tumor efficacy in PD-1-refractory tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 689.

Abstract 2964: Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042

Abstract Tumor-associated macrophages (TAMs) are the largest immune cell population in many cancers and play a key role in establishing the immunosuppressive tumor microenvironment (TME) that enables tumor progression. However, TAMs are phenotypically plastic and have the potential to be reprogrammed into immunostimulatory cells that enhance innate and adaptive anti-tumor immunity. To this end, we developed BDC-3042, an agonistic antibody targeting an immune-activating receptor expressed on TAMs known as Dectin-2 (CLEC6A). Dectin-2 is a C-type lectin receptor (CLR) known best for its role in pathogen recognition and induction of protective immune responses against fungi and other microbes. We previously demonstrated that Dectin-2 agonism with natural ligands stimulates pro-inflammatory cytokine secretion and antigen presentation by TAMs, resulting in robust CD8+ T cell-mediated anti-tumor immunity in syngeneic mouse models. Here we present our preclinical studies demonstrating the therapeutic potential of the Dectin-2 agonistic antibody, BDC-3042, as a novel TAM-directed immunotherapy for diverse human cancers. BDC-3042 exhibits strong binding to Dectin-2-expressing macrophages generated in vitro and to primary human TAMs from a range of solid tumor types. In contrast, BDC-3042 binds weakly to peripheral monocytes and minimally to other immune cells in blood and tumor tissues. Macrophages exposed to cytokines and growth factors commonly found in the TME exhibit increased Dectin-2 expression and BDC-3042 binding. Treatment with BDC-3042 activates in vitro-generated macrophages and primary TAMs to produce an array of pro-inflammatory cytokines and chemokines associated with anti-tumor immunity. Consistent with low target expression, BDC-3042 elicits little to no activation of peripheral monocytes or cells in whole blood. BDC-3042 activity is dependent on both Dectin-2 and FcγRs, as indicated through studies utilizing Fc variants with enhanced or attenuated effector function. In mice with humanized immune systems, BDC-3042 elicits activation of TAMs, as evidenced by modulation of key activation markers. Systemically administered BDC-3042 mediates anti-tumor efficacy as a monotherapy, and combination with checkpoint blockade therapy enhances anti-tumor efficacy. The data presented demonstrate the therapeutic potential of targeting Dectin-2 expressed by TAMs with the agonistic antibody BDC-3042 as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy. Citation Format: Justin A. Kenkel, Fang Xiao, Po Y. Ho, Jess L. Nolin, Rishali K. Gadkari, Laughing Bear Torrez, David T. Omstead, Katelynn A. McEachin, Jason Ptacek, Rachel Grgich, Cecelia I. Pearson, Stefan Chun, Cindy L. Kreder, Karla A. Henning, Han K. Kim, Lu Xu, Steven J. Chapin, Michael N. Alonso, Shelley E. Ackerman. Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2964.

Abstract 6663: Immunostimulatory gene therapy targeting CD40/4-1BB in combination with chemotherapy induces an inflammatory gene profile in tumors from patients with advanced disease

Abstract In the LOKON002 phase I/II clinical trial (NCT03225989), therapy with LOAd703 (delolimogene mupadenorepvec) is investigated in combination with gemcitabine-based chemotherapy in patients with advanced cancer. LOAd703 is an immunostimulatory gene therapy utilizing an oncolytic adenovirus that is engineered to express two transgenes (trimerized membrane-bound CD40L (TMZ-CD40L) and 4-1BBL), which are key players in the induction of an anti-tumor immune response. Herein, we present the preliminary immunological evaluation of the first 27 patients enrolled in the trial. The primary tumor was pancreatic cancer (n=16), colorectal cancer (n=5), ovarian cancer (n=3) or biliary cancer (n=3). Patients received a maximum of eight intratumoral LOAd703 treatments, given biweekly, and chemotherapy was administered at the same time according to standard protocol. Matched tumor biopsies from the injected tumor lesion were taken at baseline and after six LOAd703 treatments, and analyzed for gene expression with nCounter® PanCancer Immune Profiling Panel from NanoString (n=12; 7 patients analyzed so far). Blood samples were collected for isolation of serum samples and peripheral blood mononuclear cells, and these were analyzed with multiplex assays (Meso Scale Diagnostics) (n=15) and flow cytometry (n=27), respectively. Preliminary results indicate that after treatment, most tumors displayed a strong inflammatory profile with an upregulation of gene profiles, which have been shown to predict responses to immune checkpoint inhibitor therapy (T cell inflamed profile (Ayers et al.) & T effector and IFNγ associated profile (Fehrenbacher et al.)). We also noted an upregulation of genes connected to dendritic cell activation and antigen presentation (MHC class I and II, CD80, CD86, TAP1/2), as well as adenoviral-response genes. Moreover, we observed systemic treatment effects after three LOAd703 treatments, as shown by an increase in serum levels of pro-inflammatory cytokines and chemokines (IFNγ, IL-15, CXCL10, CCL2, IL-8, IL-6), as well as an increased percentage of CD8+ effector memory T cells (CD45RA-CCR7-) and PD-1+ CD8+ T cells in the blood. The fractions of natural killer (NK) cells (CD14-CD3-CD56+CD16+) and M2-like macrophages (CD11b+CD163+) were reduced in the blood at that time point. In conclusion, LOAd703 therapy in combination with chemotherapy generated an inflamed tumor microenvironment in tumors that are normally seen as immunologically “cold”. Hence, LOAd703 may be able to prime tumors for immune checkpoint inhibitors or other immunotherapies, such as adoptive T or NK cell transfer. Citation Format: Jessica Wenthe, Emma Eriksson, Sedigheh Naseri, Linda Sandin, Amanda Hahn, Sandra Irenaeus, Anders Sundin, Justyna Leja-Jarblad, Gustav Ullenhag, Tanja Lövgren, Angelica Loskog. Immunostimulatory gene therapy targeting CD40/4-1BB in combination with chemotherapy induces an inflammatory gene profile in tumors from patients with advanced disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6663.

Abstract 1852: Systemic delivery of ST317, a cell penetrating STING pathway sensitizer, results in strong anti-tumor activity

Abstract Pharmacological activation of the STING pathway is being assessed as a strategy to stimulate both the innate and adaptive arms of the immune system to trigger an anti-tumor immune response in so-called "cold" tumors. Although the first generation of STING agonists dosed intratumorally showed a strong anti-tumor response in preclinical models, positive clinical data has so far been limited, potentially due to reduced efficacy in non-injected tumors. Newer classes of STING agonists that are delivered systemically are expected to deliver more consistent and predictable levels of STING activation, but may less tolerated due to untargeted activation of the immune system resulting in immune related adverse events. We have previously demonstrated that IFI16 promotes activation of the cGAMP-activated STING pathway by a process relying on IFI16 PYRIN domain-dependent protein-protein interactions1. Based on these findings, an IFI16 Pyrin domain-derived peptide incorporating a cell penetrating peptide that recapitulates the agonist-dependent activation of the STING pathway by IFI16, was developed. This peptide, ST317, acts as a STING sensitizer for effective and safe induction of anti-tumour immune responses only at sites where STING agonists are present. In vitro, ST317 sensitizes STING to pathway activators (Cytoplasmic DNA and cGAMP) and pharmacological STING agonists including ADU-S100 and diABZI compound 3, reducing the EC50 values of agonist-induced STING pathway activation while inducing supraphysiological (i.e. increasing the EMax) levels of pro-inflammatory cytokines/chemokines (e.g. Type I interferon and CXCL10) in cell line and primary cells. Mechanistically, ST317 sensitizes STING to cGAMP by accelerating and increasing STING translocation from the endoplasmic reticulum to the Golgi, resulting in earlier and longer activation of the STING pathway components, as determined by the phosphorylation kinetics of TBK1 and IRF3, as well as STING itself. Consistent with its in vitro pharmacological profile, ST317 can be safely administered parenterally without evidence of peripheral STING activation. In vivo experiments in immunocompetent syngeneic mouse models demonstrate that ST317 dosed I.V. was well tolerated and was associated with anti-tumor efficacy when used as a single agent or in combination with immune checkpoint blockers such as anti-PD1. In addition, ST317 improved the anti-tumor response to a pharmacological STING agonist at doses that do not cause immune cell death that result from over-dosing this type of agent. In summary, ST317 represents a new class of STING pathway activator, which enables safe systemic administration while giving rise to enhanced STING pathway activation only at sites at which STING agonists are present. The data presented here warrant the assessment of ST317 in cancer patients in a clinical trial. 1. Jønsson et al. 2017 DOI: 10.1038/ncomms14391 Citation Format: Claus Elsborg Olesen, Esben Axelgaard, Emil Nilsson, Anna H. Hansen, Isis Benoit-Lizon, Justin V. Joseph, Kristina Byskov, Magnus Strandh, Maja Myrcha, Pernille Rimmer Noer, Ulrich Sensfuss, Richard Charles Sainson, Richard C. Bethell, Martin R. Jakobsen. Systemic delivery of ST317, a cell penetrating STING pathway sensitizer, results in strong anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1852.

Enhanced Valsa canker resistance conferred by expression of MdLecRK-S.4.3 in Pyrus betulifolia is largely suppressed by PbePUB36.

L-type lectin receptor-like kinases (L-LecRKs) act as sensors of extracellular signals and as initiators for plant immune responses; however, the function of LecRK-S.4 in plant immunity has not yet been extensively investigated. In the present study we found that MdLecRK-S.4.3 in apple (Malus domestica), a homologous gene of LecRK-S.4, was differentially expressed during infection by Valsa mali and Valsa pyri. Overexpression of MdLecRK-S.4.3 facilitated the induction of immune responses and enhanced the resistance to Valsa canker of fruits of apple and pear (Pyrus betulifolia), and of suspension cells of pear 'Duli-G03'. The expression of PbePUB36, a RLCK XI sub-family member, was significantly repressed in the MdLecRK-S.4.3-overexpressing cell lines. Overexpression of PbePUB36 interfered with the resistance to Valsa canker and the immune response caused by up-regulation of MdLecRK-S.4.3. In addition, we found that MdLecRK-S.4.3 interacted with BAK1 and/or PbePUB36 in vivo. Thus, whilst MdLecRK-S.4.3 activated various immune responses and positively regulated Valsa canker resistance, this could be largely compromised by PbePUB36. MdLecRK-S.4.3 interacted with PbePUB36 and/or MdBAK1 to mediate the immune responses. Our finding provides a basis for further examination of the molecular mechanisms underlying resistance to Valsa canker, and can contribute to resistance breeding.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

PD04-12 RADIOTHERAPEUTIC TREATMENT OF PROSTATE CANCER CELLS PROMOTES THE RELEASE OF POTENTIALLY DELETERIOUS AND IMMUNOLOGICALLY ACTIVE EXTRACELLULAR VESICLES

You have accessJournal of UrologyCME1 Apr 2023PD04-12 RADIOTHERAPEUTIC TREATMENT OF PROSTATE CANCER CELLS PROMOTES THE RELEASE OF POTENTIALLY DELETERIOUS AND IMMUNOLOGICALLY ACTIVE EXTRACELLULAR VESICLES Ryan Molony, Sarah Kerns, Brian Marples, Emmanuel Oshodi, YuhChyau Chen, and Yi-Fen Lee Ryan MolonyRyan Molony More articles by this author , Sarah KernsSarah Kerns More articles by this author , Brian MarplesBrian Marples More articles by this author , Emmanuel OshodiEmmanuel Oshodi More articles by this author , YuhChyau ChenYuhChyau Chen More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: One in four prostate cancer (PCa) patients will develop late hematuria and other delayed bladder toxicities following radiotherapy (RT) through poorly understood mechanisms. Membrane-enclosed extracellular vesicles (EVs) are released from tumor and non-tumor cells at baseline, and their composition can change markedly in response to stimuli. The impact of RT-induced EV (RT-EV) release from PCa cells or other cell populations in the tumor microenvironment, including immune cells, have yet to be characterized. Here, we examined the dynamics and composition of RT-EVs released by PCa cells and immune cells in response to irradiation, and their association with bladder tissue toxicity. METHODS: Urinary EV cargo proteins in pre- and post-RT PCa patient samples were analyzed and compared by LC-MS/MS. The impact of RT on EV release kinetics was analyzed by nanoparticle tracking analysis. RT-EVs and naïve EVs were collected from PCa patients and PCa cells lines (C4-2 and LNCaP). Their mechanistic effects on normal SV-HUC urothelial cells were assessed, including cellular viability, reactive oxygen species (ROS) production, and cytokine release. The effects of RT-EVs and RT on the activation of immune cell populations were monitored by treating Jurkat T cells or HL-60-derived neutrophils with RT-EVs or irradiating these cells and evaluating activation, cytokine release, and ROS production. RESULTS: The post-RT urinary EVs of a PCa patient who later developed hematuria were enriched for potentially caustic neutrophil-associated proteins relative to EVs from a hematuria-free patient. RT significantly enhanced the release of EVs from PCa cell lines and neutrophils, and these RT-EVs or direct irradiation promoted increased ROS production by HL-60 neutrophils, Jurkat T cells, and SV-HUC urothelial cells while also increasing interleukin (IL)-1β release from SV-HUC cells consistent with the induction of a damage-associated innate immune response. CONCLUSIONS: These results suggest RT can provoke the release of immunologically active and potentially deleterious EVs from PCa cells and local immune cell populations that can contribute to oxidative stress and inflammatory response induction in local and distant cell populations including neutrophils, T cells, and urothelial cells, potentially driving a feedback loop exacerbating RT-associated tissue damage. Source of Funding: University of Rochester Medical Center Departmental Funding © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e146 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryan Molony More articles by this author Sarah Kerns More articles by this author Brian Marples More articles by this author Emmanuel Oshodi More articles by this author YuhChyau Chen More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

A liposomal vaccine promotes strong adaptive immune responses via dendritic cell activation in draining lymph nodes.

Subunit proteins provide a safe source of antigens for vaccine development especially for intracellular infections which require the induction of strong cellular immune responses. However, those antigens are often limited by their low immunogenicity. In order to achieve effective immune responses, they should be encapsulated into a stable antigen delivery system combined with an appropriate adjuvant. As such cationic liposomes provide an efficient platform for antigen delivery. In the present study, we describe a liposomal vaccine platform for co-delivery of antigens and adjuvants able to elicit strong antigen-specific adaptive immune responses. Liposomes are composed of the cationic lipid dimethyl dioctadecylammonium bromide (DDAB), cholesterol (CHOL) and oleic acid (OA). Physicochemical characterization of the formulations showed that their size was in the range of ∼250 nm with a positive zeta potential which was affected in some cases by the enviromental pH facilitating endosomal escape of potential vaccine cargo. In vitro, liposomes were effectively taken up by bone marrow dendritic cells (BMDCs) and when encapsulated IMQ they promoted BMDCs maturation and activation. Upon in vivo intramuscular administration, liposomes' active drainage to lymph nodes was mediated by DCs, B cells and macrophages. Thus, mice immunization with liposomes having encapsulated LiChimera, a previously characterized anti-leishmanial antigen, and IMQ elicited infiltration of CD11blow DCs populations in draining LNs followed by increased antigen-specific IgG, IgG2a and IgG1 levels production as well as indcution of antigen-specific CD4+ and CD8+ T cells. Collectively, the present work provides a proof-of-concept that cationic liposomes composed of DDAB, CHOL and OA adjuvanted with IMQ provide an efficient delivery platform for protein antigens able to induce strong adaptive immune responses via DCs targeting and induction of maturation.

Shatavarin-IV saponin adjuvant elicits IgG and IgG2b responses against Staphylococcus aureus bacterin in a murine model

Asparagus adscendens Roxb. also known as “safed musli” or “shatavari” is a medicinal plant commonly found in South Asian countries. Shatavari is effective for the treatment of gastric ulcers, renal stones, bronchitis, diabetes, diabetic neuropathy, irritable bowel syndrome, alcohol withdrawal and has reported immunostimulatory effects. In this study, the adjuvant potential of Shatavarin-IV saponin against Staphylococcus aureus bacterin in mice was investigated. Shatavarin-IV was evaluated for its toxicity and immunomodulatory potential against S. aureus bacterin in mice. Cellular and humoral immune responses were assessed. Shatavarin-IV was isolated from the fruit extract of Asparagus adscendens. The confirmation of the isolated molecule as Shatavarin-IV was done via TLC-based comparison with the standard molecule. Further, the structure was confirmed by using extensive spectroscopic analyses and comparing the observed data with literature reports. It was found safe up to the dose of 0.1 mg in the mice model. Shatavarin-IV adjuvant elicited IgG and IgG2b responses at the dose of 40 μg against S. aureus bacterin. However, the cell-mediated immune response was lesser as compared with the commercial Quil-A saponin . We demonstrated that Shatavarin-IV saponin adjuvant produced an optimum humoral immune response against S. aureus bacterin. These results highlight the potential of Shatavarin-IV as an adjuvant in a combination adjuvant in vaccine formulations for induction of potent immune response.

Nanovaccines for cancer immunotherapy: Focusing on complex formation between adjuvant and antigen.

As an interesting cancer immunotherapy approach, cancer vaccines have been developed to deliver tumor antigens and adjuvants to antigen-presenting cells (APCs). Although the safety and easy production shifted the vaccine designing platforms toward the subunit vaccines, their efficacy is limited due to inefficient vaccine delivery. Nanotechnology-based vaccines, called nanovaccines, address the delivery limitations through co-delivery of antigens and adjuvants into lymphoid organs and APCs and their intracellular release, leading to cross-presentation of antigens and induction of potent anti-tumor immune responses. Although the nanovaccines, either as encapsulating agents or biomimetic nanoparticles, exert the desired anti-tumor activities, there is evidence that the mixing formulation to form nanocomplexes between antigens and adjuvants based on the electrostatic interactions provokes high levels of immune responses owing to Ags' availability and faster release. Here, we summarized the various platforms for developing cancer vaccines and the advantages of using delivery systems. The cancer nanovaccines, including nanoparticle-based and biomimetic-based nanovaccines, are discussed in detail. Finally, we focused on the nanocomplexes formation between antigens and adjuvants as promising cancer nanovaccine platforms.

PhaseI study of a novel therapeutic vaccine as perioperative treatment for patients with surgically resectable hepatocellular carcinoma: The YCP02 trial.

Developing effective adjuvant therapies is essential for improving the surgical outcomes in patients with hepatocellular carcinoma (HCC). Immunotherapy against HCC has become a promising strategy; however, only approximately 30% of all HCC patients respond to immunotherapy. Previously, we generated the novel therapeutic vaccine comprising multi-human leukocyte antigen-binding heat shock protein70/glypican-3 peptides with a novel adjuvant combination of hLAG-3Ig and poly-ICLC. We also confirmed the safety of this vaccination therapy, as well as its capacity for the effective induction of immune responses in a previous clinical trial. In this phaseI study, we administered this vaccine intradermally six times before surgery, and 10 times after surgery to patients with untreated, surgically resectable HCC (stage II to IVa). The primary end-points of this study were the safety and feasibility of this treatment. We also analyzed the resected tumor specimens pathologically using hematoxylin-eosin staining and immunohistochemistry for heat shock protein70, glypican3, CD8 and programmed death-1. A total of 20 human leukocyte antigen-matched patients received this vaccination therapy with an acceptable side-effect profile. All patients underwent planned surgery without vaccination-related delay. Immunohistochemical analyses revealed that potent infiltration of CD8+ Tcells into tumors with target antigen expression was observed in 12 of 20 (60%) patients. This novel therapeutic vaccine was safe as perioperative immunotherapy for patients with HCC, and has the potential to strongly induce CD8+ Tcells infiltration into tumors.