Enhanced Dendritic Cells Maturation Research Articles

Combination of MHI148 Targeted Photodynamic Therapy and STING Activation Inhibits Tumor Metastasis and Recurrence.

Metastasis and recurrence are notable contributors to mortality associated with breast cancer. Although immunotherapy has shown promise in mitigating these risks after conventional treatments, its effectiveness remains constrained by significant challenges, such as impaired antigen presentation by dendritic cells (DCs) and inadequate T cell infiltration into tumor tissues. To address these limitations, we developed a multifunctional nanoparticle platform, termed GM@P, which consisted of a hydrophobic shell encapsulating the photosensitizer MHI148 and a hydrophilic core containing the STING agonist 2'3'-cGAMP. This design elicited robust type I interferon responses to activate antitumor immunity. The GM@P nanoparticles loaded with MHI148 specifically targeted breast cancer cells. Upon exposure to 808 nm laser irradiation, the MHI148-loaded nanoparticles produced toxic reactive oxygen species (ROS) to eradicate tumor cells through photodynamic therapy (PDT). Notably, PDT stimulated immunogenic cell death (ICD) to foster the potency of antitumor immune responses. Furthermore, the superior photoacoustic imaging (PAI) capabilities of MHI148 enabled the simultaneous visualization of diagnostic and therapeutic procedures. Collectively, our findings uncovered that the combination of PDT and STING activation facilitated a more conducive immune microenvironment, characterized by enhanced DC maturation, infiltration of CD8+ T cells, and proinflammatory cytokine release. This strategy stimulated local immune responses to augment systemic antitumor effects, offering a promising approach to suppress tumor growth, inhibit metastasis, and prevent recurrence.

Rapamycin Induces Phenotypic Alterations in Oral Cancer Cells That May Facilitate Antitumor T Cell Responses.

In this study, we investigated the antitumor immunomodulatory effects of rapamycin in oral cancer. We examined the proliferation, apoptosis, and migration of cancer cells and investigated the cell surface expression levels of immune accessory molecules and T cell immune responses in vitro. We investigated the effect of in vivo administration of rapamycin on immune cell distribution and T cell immune responses in oral tumor-bearing mice. Rapamycin treatment significantly inhibited OSCC cell proliferation and migration, increased apoptotic cell death, and upregulated cell surface expression of several immune accessory and adhesion molecules, including CD40, CD83, PD-L1, PD-L2, MHC class I, P-selectin, and VCAM-1. These cancer cells augmented T cell proliferation. In vivo rapamycin administration significantly attenuated mouse tumor growth with an increased proportion of immune cells, including CD4+ T cells, CD8+ T cells, and dendritic cells (DCs); decreased the proportion of immune suppressive cells, such as myeloid-derived suppressor cells and regulatory T cells; enhanced DC maturation and upregulated the surface expression of CD40, CD86, and ICAM-1. Our results suggest that the therapeutic effect of mTOR inhibition in oral cancer can cause direct antitumor and immunomodulatory effects.

Intranasal B5 promotes mucosal defence against Actinobacillus pleuropneumoniae via ameliorating early immunosuppression

ABSTRACT Actinobacillus pleuropneumoniae (APP) is an important pathogen of the porcine respiratory disease complex, which leads to huge economic losses worldwide. We previously demonstrated that Pichia pastoris-producing bovine neutrophil β-defensin-5 (B5) could resist the infection by the bovine intracellular pathogen Mycobacterium bovis. In this study, the roles of synthetic B5 in regulating mucosal innate immune response and protecting against extracellular APP infection were further investigated using a mouse model. Results showed that B5 promoted the production of tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-β in macrophages as well as dendritic cells (DC) and enhanced DC maturation in vitro. Importantly, intranasal B5 was safe and conferred effective protection against APP via reducing the bacterial load in lungs and alleviating pulmonary inflammatory damage. Furthermore, in the early stage of APP infection, we found that intranasal B5 up-regulated the secretion of TNF-α, IL-1β, IL-17, and IL-22; enhanced the rapid recruitment of macrophages, neutrophils, and DC; and facilitated the generation of group 3 innate lymphoid cells in lungs. In addition, B5 activated signalling pathways associated with cellular response to IFN-β and activation of innate immune response in APP-challenged lungs. Collectively, B5 via the intranasal route can effectively ameliorate the immune suppression caused by early APP infection and provide protection against APP. The immunization strategy may be applied to animals or human respiratory bacterial infectious diseases. Our findings highlight the potential importance of B5, enhancing mucosal defence against intracellular bacteria like APP which causes early-phase immune suppression.

Engineered Microcystis aerugiosa Hydrogel as an Anti‐Tumor Therapeutic by Augmenting Tumor Immunogenicity and Immune Responses

AbstractCancer immunotherapy holds great promise but is generally limited by insufficient induction of anticancer immune responses. In this work, Microcystis aerugiosa (MA) is observed to act as an immune stimulator, which activates the cGAS‐STING and IRF‐signaling pathways of dendritic cells (DCs), induces immunostimulatory factors production, eventually resulting in the death of tumor cells by promoting cytotoxic CD8+ T cells infiltration. To further enhance the tumor immunogenicity, MA is engineered with polydopamine (PDA) and assembled in injectable Pluronic F127 hydrogels (denoted as MA@PDA‐F127). The introduction of PDA endows the MA@PDA‐F127 with photothermal therapy capability, which enhances the immunogenicity by in situ exposing damage‐associated molecular patterns and tumor antigens from dying tumor cells. Importantly, the surface‐rich reaction sites in thermosensitive MA@PDA‐F127 hydrogels capture antigens, facilitating long retention of antigens exposure and enhancing DCs maturation for advanced anti‐tumor immune response. Accordingly, in situ administration of MA@PDA‐F127 hydrogels in a single dose achieves efficient eradication of both primary and distant tumors in an orthotopic tumor metastasis model. Furthermore, this hydrogel can sensitize immune checkpoint inhibitor therapy and enhance T cell infiltration in a murine tumor model. Collectively, MA@PDA‐F127 hydrogel offers an effective anti‐tumor therapeutic by augmenting tumor immunogenicity and activating a systematic immune response.

Interferon-beta mediated STAT4up-regulation are distinct in the ex vivohuman CD4 +T-cells priming dendritic cells

Abstract STAT4 (Signal Transducer Activator of Transcription factor 4) polymorphisms are risk factors contributed to autoimmune disease. While STAT4 has a dominant role in promoting interferon-gamma (IFN-g) production in T and NK cells, its existence and function exert in human dendritic cells (DCs) are still unknown. To characterize the STAT4 expression in human peripheral DCs, we isolated DCs from buffy coat of health blood donors (n = 4). The ex vivo DCs were enriched and sorted into 3 subtypes including plasmacytoid DCs (pDCs, CD123 +), CD8 +T-cells priming DC (cDC1, CLEC9A +), and CD4 +T-cells priming DC (cDC2/3, CD1c +) through negative selection bead and flow cytometry. The presence of STAT4 as well as its potential target genes (interleukin-12, IL-12 and interleukin-23, IL-23) expression were examined in IFN-beta induced DCs maturation compared to immature DCs, using Real-Time PCR. We found the proportion of DCs subsets in peripheral mononuclear cells which are 0.05%, 0.02% and 0.3% for pDC, cDC1 and cDC2/3, respectively. The IFN-beta enhanced DC maturation markers (HLA-DR +CD86 +), especially in cDC2/3. Interestingly, up-regulated STAT4 expression (2.5-fold) were significantly prominent in mature cDC2/3 compared to immature (p < 0.01), while IFN-beta showed no influence on STAT4 expression in the cDC1 and pDC (p > 0.05). Interestingly, elevated IL-12 and IL-23 expression were significantly observed in activated cDC2/3 (p < 0.05). Further study to investigate the role of STAT4 in human cDC2/3 are needed. Our result firstly discover the existing of STAT4 expression in the ex vivo peripheral human DC. Supported by Chulalongkorn University (Fundamental Fund, CU_FRB65_hea (84)_179_37_09).

Manganese-phenolic nanoadjuvant combines sonodynamic therapy with cGAS-STING activation for enhanced cancer immunotherapy

Dendritic cells (DCs) play a key role in the presentation of tumor antigens to initiate innate and adaptive immune responses. While the tumor microenvironment (TME) contains a network of immunosuppressive factors that inhibit the differentiation and maturation of DCs. Immunoadjuvants have been focusing on modulating DCs function to improve cancer immunotherapy. Herein, a phenolic nanoadjuvant was developed by self-assembly of the sonosensitizer polymer (PEG-b-IR), GSH inhibitor (sabutoclax), Mn2+ and TME acidic sensitive phenolic polymer (PEG-b-Pho) via metal–phenolic coordination. The combinational action of SDT-mediated ICD effect and Mn2+ promoted the activation of the cGAS-STING pathway significantly enhanced DCs maturation. Furthermore, this phenolic nanoadjuvant greatly sensitizes tumors to PD-L1 checkpoint blockade immunotherapy, resulting in efficiently inhibiting the growth of distant tumors and restraining lung metastasis. Therefore, our work provides the potential to remedy the therapeutic limitation of insufficient antitumor immunity for enhanced cancer immunotherapy.

Engineered porous/hollow Burkholderia pseudomallei loading tumor lysate as a vaccine

Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and adjuvant CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8+-induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.

Methotrexate enhances antigen presentation and maturation of tumour antigen-loaded dendritic cells through NLRP3 inflammasome activation: a strategy for dendritic cell-based cancer vaccine.

Background:Dendritic cells (DCs) are antigen-presenting cells that play a pivotal role in adaptive cell-mediated immunity by priming and activating T cells against specific tumour and pathogenic antigens. Methotrexate (MTX), a folate derivative, functions as an immunoregulatory agent. However, the possible effect of MTX on tumour antigen-loaded DCs has not yet been investigated.Methods:We analysed the effect of MTX on the maturation and function of DCs along with tumour cell lysates (TCLs). Using bone marrow-derived DCs, we investigated the effect of MTX combined TCL-loaded DCs on T cells priming and proliferation. We also tested the anti-tumour immune effect on DCs when treated with MTX and/or TCL in vivo.Results:MTX combined with TCL not only enhanced DC maturation and stimulated cytokine release but also promoted CD8+ T cell activation and proliferation. The latter was associated with increased tumour antigen uptake and cross-presentation to T cells. Mechanistically, DC maturation and antigen presentation were partly modulated by NLRP3 inflammasome activation. Furthermore, immunisation of mice with MTX and TCL-pulsed DCs before a tumour challenge significantly delayed tumour onset and retarded its growth. This protective effect was due to priming of IFN-γ releasing CD8+ T cells and enhanced killing of tumour cells by cytotoxic T lymphocytes isolated from these immunised mice.Conclusion:MTX can function as a potent adjuvant in DC vaccines by increasing antigen presentation and T cell priming. Our findings provide a new strategy for the application of DC-based anti-tumour immunotherapy.

Immunomodulatory activity of IR700-labelled affibody targeting HER2

There is an urgent need to develop therapeutic approaches that can increase the response rate to immuno-oncology agents. Photoimmunotherapy has recently been shown to generate anti-tumour immunological responses by releasing tumour-associated antigens from ablated tumour cell residues, thereby enhancing antigenicity and adjuvanticity. Here, we investigate the feasibility of a novel HER2-targeted affibody-based conjugate (ZHER2:2395-IR700) selectively to induce cancer cell death in vitro and in vivo. The studies in vitro confirmed the specificity of ZHER2:2395-IR700 binding to HER2-positive cells and its ability to produce reactive oxygen species upon light irradiation. A conjugate concentration- and light irradiation-dependent decrease in cell viability was also demonstrated. Furthermore, light-activated ZHER2:2395-IR700 triggered all hallmarks of immunogenic cell death, as defined by the translocation of calreticulin to the cell surface, and the secretion of ATP, HSP70/90 and HMGB1 from dying cancer cells into the medium. Irradiating a co-culture of immature dendritic cells (DCs) and cancer cells exposed to light-activated ZHER2:2395-IR700 enhanced DC maturation, as indicated by augmented expression of CD86 and HLA-DR. In SKOV-3 xenografts, the ZHER2:2395-IR700-based phototherapy delayed tumour growth and increased median overall survival. Collectively, our results strongly suggest that ZHER2:2395-IR700 is a promising new therapeutic conjugate that has great potential to be applicable for photoimmunotherapy-based regimens.

Combined hybrid structure of siRNA tailed IVT mRNA (ChriST mRNA) for enhancing DC maturation and subsequent anticancer T cell immunity

RNA therapeutics have received much attention in the development of anti-cancer therapies. Among them, synthetic mRNA (IVT mRNA) was investigated for cancer immunotherapy due to its abilities to express tumor associated antigens with stimulation of immune responses in dendritic cells (DCs). Despite of its great potential, several hurdles were remained such as insufficient immune stimulation and DC maturation. In this study, we aimed to present a novel IVT mRNA that can simultaneously express tumor associated antigens while suppress STAT3 proteins. Combined functions of siRNA and IVT mRNA were investigated and the hybrid structure of siRNA tailed mRNA (ChriST mRNA) was developed. We prepared the ChriST mRNA by employing polyA tail structures with RNAi sequences at the 3′ end of mRNA. Complementary strands were annealed to form duplex siRNA structure to induce STAT3 gene silencing. In addition, a hybrid structure of DNA/RNA was introduced into the ChriST mRNA between polyA tail and RNAi sequences. It was expected that DNA/RNA duplex would be readily cleaved by RNase H in the intracellular environment. After the cleavage, ChriST mRNA was fully functionalized in cells and exhibited enhanced tumor specific DC maturation.

Immunostimulatory Potential of MoS2 Nanosheets: Enhancing Dendritic Cell Maturation, Migration and T Cell Elicitation.

BackgroundDue to their extraordinary physical and chemical properties, MoS2 nanosheets (MSNs) are becoming more widely used in nanomedicine. However, their influence on immune systems remains unclear.Materials and MethodsTwo few-layered MSNs at sizes of 100–250 nm (S-MSNs) and 400–500 nm (L-MSNs) were used in this study. Bone marrow-derived dendritic cells (DCs) were exposed to both MSNs at different doses (0, 8, 16, 32, 64, 128 µg/mL) for 48 h and subjected to analyses of surface marker expression, cytokine secretion, lymphoid homing and in vivo T cell priming.ResultsDifferent-sized MSNs of all doses did not affect the viability of DCs. The expression of CD40, CD80, CD86 and CCR7 was significantly higher on both S-MSN- and L-MSN-treated DCs at a dose of 128 μg/mL. As the dose of MSN increased, the secretion of IL-12p70 remained unchanged, the secretion of IL-1β decreased, and the production of TNF-α increased. A significant increase in IL-6 was observed in the 128 µg/mL L-MSN-treated DCs. In particular, MSN treatment dramatically improved the ex vivo movement and in vivo homing ability of both the local resident and blood circulating DCs. Furthermore, the cytoskeleton rearrangement regulated by ROS elevation was responsible for the enhanced homing ability of the MSNs. More robust CD4+ and CD8+ T cell proliferation and activation (characterized by high expression of CD107a, CD69 and ICOS) was observed in mice vaccinated with MSN-treated DCs. Importantly, exposure to MSNs did not interrupt LPS-induced DC activation, homing and T cell priming.ConclusionFew-layered MSNs ranging from 100 to 500 nm in size could play an immunostimulatory role in enhancing DC maturation, migration and T cell elicitation, making them a good candidate for vaccine adjuvants. Investigation of this study will not only expand the applications of MSNs and other new transition metal dichalcogenides (TMDCs) but also shed light on the in vivo immune-risk evaluation of MSN-based nanomaterials.

The hydrogel‐encapsulated liver X receptor ligand, T0901317, enhances its antitumorigenic effects but eliminates lipogenesis

The anti‐tumorigenic functions of liver X receptor (LXR) ligands have been confirmed in several types of tumors. We previously reported that LXR ligand, T0901317 (T317), inhibited growth of inoculated LLC1 carcinoma and urethane‐induced pulmonary carcinomas by activating IFN‐γ expression in tissues, particularly in macrophages. However, T317 activates fatty acid synthesis to result in severe hepatic lipogenesis and fatty liver. Tissue‐specific activation of LXR, such as in antigen‐presenting cells (APCs) but not in liver, by ligand can enhance inhibition of tumor growth without activation of lipogenesis. Injected naphthylacetic acid modified D‐glycine‐phenylalanine‐phenylalanine‐tyrosine tetra‐peptide (GFFY) can be selectively delivered to APCs, macrophages and dendritic cells (DCs). In this study, we encapsulated T317 with GFFY hydrogel (GFFY‐T317) and determined its effect on tumor growth and hepatic lipogenesis. Compared with oral administration of T317, subcutaneous injection of GFFY‐T317 more potently inhibited LLC1 tumor growth in mice. The inhibition was dependent on induction of IFN‐γ expression in APCs by activating LXR. We further determined that GFFY‐T317 increased M1 type macrophages while reducing M2 cells in the tumor. Meanwhile, GFFY‐T317 enhanced DC maturation and infiltration to tumors. Both CD3+ and CD8+ cells in tumors were also increased, which was associated with activation of IFN‐γ expression. In contrast, tumor angiogenesis was clearly inhibited by GFFY‐T317 in an IFN‐γ‐dependent manner. In addition, GFFY‐T317 had more potent inhibitory effects on urethane‐induced lung tumor than oral administration of T317. More importantly, in these two tumor models, compared with severe liver steatosis by oral administration of T317, injection of GFFY‐T317 had little effect on lipid synthesis/metabolism in the liver, while eliminating T317‐induced liver injury. Taken together, our study demonstrates that the hydrogel‐encapsulated T317 can inhibit lung tumor growth without adverse effect of lipogenesis, indicating the hydrogel‐encapsulated LXR ligand might be a novel therapy for tumor treatment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Human Papillomavirus 16 L1 Pentamer-Loaded Hybrid Particles Vaccine System: Influence of Size on Immune Responses.

Cervical cancer remains the second-most prevalent female malignancy around the world, leading to a great majority of cancer-related mortality that occurs mainly in developing countries. Developing an effective and low-cost vaccine against human papillomavirus (HPV) infection, especially in medically underfunded areas, is urgent. Compared with vaccines based on HPV L1 viruslike particles (VLPs) in the market, recombinant HPV L1 pentamer expressed in Escherichia coli represents a promising and potentially cost-effective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have shown great potential compared to conventional aluminum salts adjuvant and is urgently needed for HPV L1 pentamer vaccines. It is well-reported that particle sizes are crucial in regulating immune responses. Nevertheless, reports on the relationship between the particulate size and the resultant immune response have been in conflict, and there is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Here, we fabricated HPV 16 L1 pentamer-loaded poly(d,l-lactide- co-glycolide) (PLGA)/lecithin hybrid particles with uniform sizes (0.3, 1, and 3 μm) and investigated the particle size effects on antigen release, activation of lymphocytes, dendritic cells (DCs) activation and maturation, follicular helper CD4+ T (TFH) cells differentiation, and release of pro-inflammatory cytokines and chemokines. Compared with the other particle sizes, 1 μm particles induced more powerful antibody protection and yielded more persistent antibody responses, as well as more heightened anamnestic responses upon repeat vaccination. The superior immune responses might be attributed to sustainable antigen release and robust antigen uptake and transport and then further promoted a series of cascade reactions, including enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in draining lymph nodes (DLNs). Here, a powerful and economical platform for HPV vaccine and a comprehensive understanding of particle size effect on immune responses for HPV L1 pentamer-based candidate vaccines are provided.

A cycle involving HMGB1, IFN-γ and dendritic cells plays a putative role in anti-tumor immunity

An important subset in regulating antitumor immunity is the maturation and accumulation of intratumor dendritic cells (DCs), inducing potent T cell cytotoxicity. In this study, we explored how the soluble abundant high-mobility group box 1 protein (HMGB1) affected DC activation and retention within lung cancers, and in which way the resultant interferon-γ (IFN-γ) further enhanced DC maturation and accumulation. It was discovered that HMGB1 was correlated with DC markers HLA-DR and CD86 in lung cancers at both mRNA and protein level. Further analyses showed HMGB1 enhanced the maturation of DCs, indicated by upregulated IFN-γ in CD8+ T cells. Additionally, HMGB1 increased the accumulation of DCs by promoting CCR5 and CXCR3 production. Moreover, the resultant IFN-γ elevated the levels of HMGB1 and DC-associated chemokines, CCL5, CXCL10 and CXCL11 in tumor cells. Hence, the HMGB1-IFN-γ cycle may represent an important mechanism underlying DC-mediated anti-tumor immune response.

Ibrutinib has a novel immunomodulatory effect by enhancing DC maturation both in vivo and in a model of inflammation

Abstract Myeloid derived suppressor cells (MDSCs) can inhibit T cell activation by altering the characteristics and activation of dendritic cells (DCs). We had previously reported that Ibrutinib enhances the expression of co-stimulatory molecules on DCs and promotes DC-mediated T cell activation. In the present study we further determined the mechanisms of DC immunomodulation by Ibrutinib. We evaluated the effect of Ibrutinib on (i) DC maturation and migration from the injection site to draining lymph nodes, (ii) T cell proliferation in vivo, and (iii) monocyte maturation to DCs using a mouse inflammation model. DCs were generated from mouse bone marrow-derived cells in the presence of vehicle or 10μM lbrutinib. CFES-labelled DCs were injected subcutaneously into the footpads of naive mice, and their migration to the regional lymph nodes and maturation profile was analyzed by flow cytometry. To evaluate T cell priming, CFSE-labelled OVA-specific T cells isolated from OT-II mice were injected into the tail vein of syngeneic mice that were prior injected with mature DCs loaded with OVA peptide. After four days, T cell proliferation was evaluated by CFSE dilution. In the inflammation model, C57BL6 mice were treated with vehicle or 6mg/kg/day Ibrutinib orally for a week. A sub-lethal dose of LPS was injected in the dorsal skin and the frequency of inflammatory monocytes and mature DCs was determined. We found that Ibrutinib significantly improves DC maturation and migration, inducing T cell activation and proliferation in vivo. In addition, Ibrutinib enhances monocyte maturation to DCs in the skin. The results suggest a novel effect of Ibrutinib, which may have therapeutic use for cancers characterized by a high MDSC infiltration.

Oncolysate-loaded Escherichia coli bacterial ghosts enhance the stimulatory capacity of human dendritic cells.

The natural adjuvant properties of bacterial ghosts (BGs) lie within the presence of intact pathogen-associated molecular patterns on their surface. BGs can improve the direct delivery, natural processing and presentation of target antigens within dendritic cells (DCs). Moreover, sensitization of human DCs by cancer cell lysate (oncolysate)-loaded BGs in the presence of IFN-α and GM-CSF enhanced DC maturation as indicated by an increased expression of maturation markers and co-stimulatory molecules, higher production of IL-12p70 and stimulation of significantly increased proliferation of both autologous CD4+ and CD8+ T cells compared to DCs matured in the presence of purified lipopolysaccharide. The induced T cells efficiently recognized oncolysate-derived tumor-associated antigens expressed by cancer cells used for the production of oncolysate. Our optimized one-step simultaneous antigen delivery and DC maturation-inducing method emerges as a promising tool for the development and implementation of next-generation cellular cancer immunotherapies.

Maturation of dendritic cells by pullulan promotes anti-cancer effect.

Previous studies have demonstrated that pullulan, a polysaccharide purified from Aureobasidium pullulans, has immune-stimulatory effects on T and B cells. Moreover, pullulan has been used as a carrier in the delivery of the antigen (Ag) peptide to lymphoid tissues. However, the in vivo effect of pullulan on dendritic cells (DC) has not been well characterized. In this study, we assessed the effect of pullulan on DC activation and anti-cancer immunity. The results showed that the pullulan treatment up-regulated co-stimulatory molecule expression and enhanced pro-inflammatory cytokine production in bone marrow-derived DCs (BMDC) in vitro and in spleen DCs in vivo. Moreover, the combination of ovalbumin (OVA) and pullulan induced OVA antigen-specific T cell activations in vivo. In tumor-bearing mice, pullulan induced the maturation of DCs in spleen and tumor draining lymph node (drLN), and promoted the OVA-specific T cell activation and migration of the T cells into the tumor. In addition, the combination of OVA and pullulan inhibited B16-OVA tumor growth and liver metastasis. The combination of tyrosinase-related protein 2 (TRP2) peptide and pullulan treatment also suppressed B16 melanoma growth. Thus, the results demonstrated that pullulan enhanced DC maturation and function, and it acted as an adjuvant in promoting Ag-specific immune responses in mice. Thus, pullulan could be a new and useful adjuvant for use in therapeutic cancer vaccines.

Abstract 297: Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and control tumor progression in a pancreas cancer model

Abstract We hypothesized that an immunostimulatory oncolytic virus (LOAd703) is a potent inducer of anti-tumor immunity and tested its biological functions in models of pancreas cancer. LOAd703 is a double-armed oncolytic adenovirus that introduces the simultaneous expression of a trimerized membrane-bound CD40L and 4-1BBL locally in the tumor aiming to induce potent Th1-mediated anti-tumor immunity. By utilizing an oncolytic adenovirus serotype 5/35 for gene transfer, virally induced tumor cell oncolysis will provide a broad release of antigens at the site of immune activation. Transgene expression is driven by a separate promoter to allow for efficient expression in both tumor cells and tumor stroma while virus replication is restricted to tumor cells by E1A delta24 deletion. CD40 ligand is a potent stimulator of myeloid cells including dendritic cells (DCs) that in turn induce robust T cell responses. CD40L can also reduce the levels of myeloid suppressor cells and M2 macrophages as well as enhancing T cell infiltration into tumors. Further, CD40-mediated signaling combined with TLR stimuli (such as adenovirus) induces intense Th1 immunity. 4-1BBL is known to provide expansion and survival signaling to pre-activated T- and NK cells. Pancreatic cancer cell lines and healthy exocrine pancreas cells were transduced with LOAd703 and it efficiently killed tumor cells while normal cells remained intact as evaluated by an MTS assay. CD40L and 4-1BBL were highly expressed by the transduced tumor cells as detected by flow cytometry. Cell lysis by LOAd703 was equally efficient as a similar oncolytic virus without transgenes demonstrating that the double transgene expression did not interfere with viral replication. Repeated (6x) peritumoral injections of LOAd703 in a xenograft Nu/Nu/Panc01 model showed efficient tumor cell growth control and sustained complete responses. The effect could be further enhanced by gemcitabine in both xenograft mice and in a syngeneic immunocompetent C57BL/6/Panc02 model. Transduction of human monocyte-derived immature DCs resulted in a strong CD40L and 4-1BBL expression without lysis of the cells. Instead, the transduced DCs matured as shown by high CD83 and IL12 expression. The addition of 4-1BBL significantly enhanced DC maturation compared to a virus containing only CD40L, or no transgenes, by expressing higher levels of CD70, IL12, TNFa, IFNg and IL21. Further, LOAd703-transduced DCs pulsed with pp65 CMV peptides potently expanded antigen-specific T cells as well as NK cells. In conclusion, LOAd703 is a novel, double-armed immunostimulatory oncolytic gene therapy that initiates robust Th1 immunity, and eradicates pancreatic cancer in experimental models. A clinical trial using LOAd703 for pancreatic cancer is underway. Citation Format: Emma Svensson, Ioanna Milenova, Rafael Moreno, Ramon Alemany, Angelica Loskog. Immunotherapy with a CD40L/4-1BBL double-armed oncolytic adenovirus drives Th1 immunity and control tumor progression in a pancreas cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 297. doi:10.1158/1538-7445.AM2015-297

Immunoadjuvant activity of the nanoparticles’ surface modified with mannan

Mannan (MN) is the natural ligand for mannose receptors, which are widely expressed on dendritic cells (DCs). The purpose of this study was to assess the effect of formulation parameters on the immunogenicity of MN-decorated poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) in terms of their ability to stimulate DC phenotypic as well as functional maturation. For this purpose, NPs were formulated from either ester-terminated or COOH-terminated PLGA. Incorporation of MN in NPs was achieved through encapsulation, physical adsorption or chemical conjugation. Murine bone marrow derived DCs (BMDCs) were treated with various NP formulations and assessed for their ability to up-regulate DC cell surface markers, secrete immunostimulatory cytokines and to activate allogenic T cell responses. DCs treated with COOH-terminated PLGA-NPs containing chemically conjugated MN (MN-Cov-COOH) have shown superior performance in improving DC biological functions, compared to the rest of the formulations tested. This may be attributed to the higher level of MN incorporation in the former formulation. Incorporation of MN in PLGA NPs through chemical conjugation can lead to enhanced DC maturation and stimulatory function. This strategy may be used to develop more effective PLGA-based vaccine formulations.

Sphingosine Analog AAL-R Promotes Activation of LCMV-Infected Dendritic Cells

Sphingosine analogs display diverse immunoregulatory activities with curative potential in autoimmune diseases and viral infections. Recently, the sphingosine analog AAL-R was shown to increase DC activation upon TLR7 stimulation. Here, we investigated the effect of AAL-R on activation of dendritic cells (DCs) infected by lymphocytic choriomeningitis virus (LCMV). Concomitant treatment of LCMV-infected DCs with AAL-R enhanced DC maturation and DC ability to stimulate and expand antiviral CD8(+) T cells. Importantly, AAL-R's stimulatory activity was abrogated in type I interferon (IFN) receptor-deficient DCs following LCMV infection. In support of this observation, AAL-R increased type I IFN production from DCs infected with LCMV. Taken together, the sphingosine analog could directly act on DCs to promote defensive host DC responses to the viral invasion via type I IFN signaling.