Immune-stimulatory Function Research Articles

Viscum album (mistletoe) extract for dogs with cancer?

Compared with the options available to human patients with cancer, treatment choices for dogs are often more limited. Chemotherapy is frequently the first-line treatment for many cancers. However, its efficacy can be limited, and its side effects can affect the quality of the remaining life. This paper briefly summarizes the experience with Viscum album L. (mistletoe) extract in human patients as a stipulation to consider treatment with mistletoe extract for canines with cancer. The mistletoe extract contains -among others - lectins and viscotoxins that have documented anti-proliferative effect on cancer cells as well as immune-stimulatory function. Importantly, it also improves the well-being of patients with cancer due to its lectin ML-1 content, which can trigger the release of endorphins. Being cross-reactive with canine cells and having a relatively low side effect profile, it raises the question of whether mistletoe preparations might be considered as part of the treatment approach for dogs with cancer.

Chlorella vulgaris Modulates Gut Microbiota and Induces Regulatory T Cells to Alleviate Colitis in Mice.

Chlorella vulgaris (C. vulgaris) is unicellular green algae consumed worldwide as a functional food. The immune stimulatory function of C. vulgaris is known; however, no study has elucidated its immune regulatory potential and associated microbiome modulation. In the current study, we aimed to validate the immune regulatory role of C. vulgaris mediated through two mechanisms. Initially, we assessed its ability to promote the expansion of the regulatory T cell (Treg) population. Subsequently, we investigated its impact on gut microbiota composition and associated metabolites. The supplementation of C. vulgaris altered the gut microbiota composition, accompanied by increased short-chain fatty acid (SCFAs) production in mice at homeostasis. We later used C. vulgaris in the treatment of a DSS-induced colitis model. C. vulgaris intervention alleviated the pathological symptom of colitis in mice, with a corresponding increase in Treg levels. As C. vulgaris is a safe and widely used food supplement, it can be a feasible strategy to instigate cross-talk between the host immune system and the intestinal flora for the effective management of inflammatory bowel disease (IBD).

EXTH-42. DUAL ROLES OF PKR ORCHESTRATE ONCOLYTIC HSV SENSITIVITY AND ANTIGEN-SPECIFIC T CELL EXPANSION

Abstract The host immune system has developed many mechanisms to defend against and clear virus infections, including PKR, cGAS-STING and TLR-MyD88 signaling pathways. Compromised anti-viral mechanisms in tumor cells allows for infection, replication and lysis by oncolytic viruses. However, compromised antiviral mechanisms, together with immune cells and the tumor microenvironment (TME), limit the sensitivity of oncolytic virus in tumor cells. We generated a novel oncolytic Herpes simplex virus, oHSV-shPKR which disables tumor-intrinsic PKR signaling. oHSV-shPKR significantly increases oHSV infection and lysis in both oncolytic virus-resistant and sensitive glioblastomas (GBMs). Infection of oHSV-shPKR in GBMs induces G2/M cell cycle arrest and inhibits tumor cell growth. oHSV-shPKR increases activation of antigen presentation cells through type I interferon signaling activation and its immune-stimulatory function to increase tumor-antigen specific CD8 T-cell expansion, including cytotoxic T lymphocytes. Preclinical studies showed that oHSV-shPKR intra-tumoral injection significantly inhibits human GBM patient derived xenograft (PDX) tumor growth in immune-deficient NSG mice and murine orthotopic GBM tumor growth in immunocompetent mice. The results demonstrate that the novel oHSV-shPKR has the potential to be used in clinical applications for both oHSV-resistant and sensitive GBM treatment.

Optineurin modulates the maturation of dendritic cells to regulate autoimmunity through JAK2-STAT3 signaling

Optineurin (OPTN) has important functions in diverse biological processes and diseases, but its effect on dendritic cell (DC) differentiation and functionality remains elusive. Here we show that OPTN is upregulated in human and mouse DC maturation, and that deletion of Optn in mice via CD11c-Cre attenuates DC maturation and impairs the priming of CD4+ T cells, thus ameliorating autoimmune symptoms such as experimental autoimmune encephalomyelitis (EAE). Mechanistically, OPTN binds to the JH1 domain of JAK2 and inhibits JAK2 dimerization and phosphorylation, thereby preventing JAK2-STAT3 interaction and inhibiting STAT3 phosphorylation to suppress downstream transcription of IL-10. Without such a negative regulation, Optn-deficient DCs eventually induce an IL-10/JAK2/STAT3/IL-10 positive feedback loop to suppress DC maturation. Finally, the natural product, Saikosaponin D, is identified as an OPTN inhibitor, effectively inhibiting the immune-stimulatory function of DCs and the disease progression of EAE in mice. Our findings thus highlight a pivotal function of OPTN for the regulation of DC functions and autoimmune disorders.

Functional Analysis of Immune Signature Genes in Th1* Memory Cells Links ISOC1 and Pyrimidine Metabolism to IFN-γ and IL-17 Production.

CCR6+CXCR3+CCR4-CD4+ memory T cells, termed Th1*, are important for long-term immunity to Mycobacterium tuberculosis and the pathogenesis of autoimmune diseases. Th1* cells express a unique set of lineage-specific transcription factors characteristic of both Th1 and Th17 cells and display distinct gene expression profiles compared with other CD4+ T cell subsets. To examine molecules and signaling pathways important for the effector function of Th1* cells, we performed loss-of-function screening of genes selectively enriched in the Th1* subset. The genetic screen yielded candidates whose depletion significantly impaired TCR-induced IFN-γ production. These included genes previously linked to IFN-γ or M. tuberculosis susceptibility and novel candidates, such as ISOC1, encoding a metabolic enzyme of unknown function in mammalian cells. ISOC1-depleted T cells, which produced less IFN-γ and IL-17, displayed defects in oxidative phosphorylation and glycolysis and impairment of pyrimidine metabolic pathway. Supplementation with extracellular pyrimidines rescued both bioenergetics and IFN-γ production in ISOC1-deficient T cells, indicating that pyrimidine metabolism is a key driver of effector functions in CD4+ T cells and Th1* cells. Results provide new insights into the immune-stimulatory function of ISOC1 as well as the particular metabolic requirements of human memory T cells, providing a novel resource for understanding long-term T cell-driven responses.

Therapeutic Value of Medicinal Mushroom Agaricus blazei Murill

Agaricus blazei Murill (AbM) is a mushroom that has been utilized in alternative drug to anticipate cardiac disease, diabetes, arthritis, increased cholesterol, cancer, hepatitis and cancers. It contains β-glucan, which contributes to decreasing blood sugar in-vivo and acts against cancer. The extract of this mushroom exerts antioxidant action in-vivo. AbM contains agaritine that exerts antitumor effects against leukemic cells in vitro. This mushroom is also found to have immune-stimulatory function, tumor growth suppression, action against allergy, antiviral effects, antimicrobial function and immune modulatory effects. Researchers also studied its action in decreasing blood cholesterol. According to several studies, AbM containing (1→6)-β-D-glucan-exerts anti angiogenesis action. The purpose of this extensive review on the medicinal value of AbM mushroom was to highlight its significance and its traditional uses by scientific evidence to determine the effectiveness of the mushroom in various evidence-based uses. Google Scholar and PubMed search engines were used to browse articles from 1994-2019 on the therapeutic value of AbM. Initially, 74 articles were found related to the therapeutic value of AbM. After reviewing the available article, 42 were selected based on the medicinal uses of AbM. It was concluded that AbM possessed various bioactive compounds that are responsible for its therapeutic effects.

Abstract 2267: MEDI5083, a novel CD40L-Fc fusion protein, activates the CD40 pathway on antigen-presenting cells and promotes robust anti-tumor immune responses in both hot and cold murine tumor models

Abstract Background: While diverse strategies may boost anti-tumor immunity, of significant interest are therapeutic approaches that are efficacious in tumors with high and low immune cell infiltration. Targeting CD40 with its natural ligand (CD40L) augments the immune-stimulatory function of antigen-presenting cells and enhances T cell priming, making this an attractive means of promoting anti-tumor responses. MEDI5083 is a novel fusion protein containing a hexameric recombinant human CD40L structure covalently linked to human IgG4p Fc. Here, we present preclinical functional characterization of MEDI5083. Methods MEDI5083 functionality was first established using several in vitro approaches with human cell lines and primary human cells. To examine anti-tumor activity in vivo, we constructed a murine surrogate (mCD40L-Fc) with similar physical and target-binding properties to those of MEDI5083. The effects of mCD40L-Fc on immune cell activation and tumor growth were determined in the immunologically cold B16F10 model of melanoma and the immunologically hot CT26 model of colon carcinoma. Results: MEDI5083 specifically bound to human CD40, triggered its internalization, and robustly stimulated CD40 signaling as measured by NF-κB activation in Ramos and THP-1 cells. MEDI5083 potently upregulated expression of MHCII and the co-stimulatory molecules CD80 and CD86 on primary human B cells and myeloid cells, while inducing secretion of pro-inflammatory cytokines. MEDI5083 also enhanced secretion of IFN-γ by memory CD8+ T cells in an antigen-recall assay. In the B16F10 model, repeated mCD40L-Fc dosing significantly decreased tumor growth. Consistent with enhancing immune cell activation and T cell priming, mCD40L-Fc treatment elevated serum levels of IL-12 and IFN-γ, and increased numbers of tumor-infiltrating CD8+ T cells. While this model responds poorly to checkpoint blockade, co-administration of mCD40L-Fc with anti-PD-L1 antibody demonstrated additivity, consistent with PD-1/PD-L1-mediated regulation of mCD40L-Fc-driven adaptive immunity. Repeated dosing of mCD40L-Fc in the CT26 model inhibited tumor growth. Treatment was associated with diminished tumor-infiltrating Treg numbers, increased CD8+ T cell:Treg ratios, and phenotypic changes in tumor-infiltrating immune cells. Tumor supernatants exhibited increased IFN-γ, consistent with enhanced anti-tumor immunity. Rechallenge of mice with complete tumor regression confirmed generation of durable memory. Conclusions: We have demonstrated that stimulation of CD40 signaling with MEDI5083 or mCD40L-Fc leads to robust activation of immune cells and enhanced adaptive immune responses. In vivo, this translates to enhanced control of both poorly- and well-infiltrated syngeneic tumors and generation of durable memory in mice with complete tumor regression. Citation Format: Deepali Malhotra, Sean Turman, Kelly McGlinchey, Yaya Wang, Yue Wang. MEDI5083, a novel CD40L-Fc fusion protein, activates the CD40 pathway on antigen-presenting cells and promotes robust anti-tumor immune responses in both hot and cold murine tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2267.

CD40L membrane retention enhances the immunostimulatory effects of CD40 ligation

In carcinomas, the nature of CD40 ligand shapes the outcome of CD40 ligation. To date, the consequences of membrane-bound CD40L (mCD40L) on its immune-stimulatory function are unknown. Here, we examined the impact of mCD40L versus soluble CD40L (sCD40L) on T24 bladder carcinoma gene expression profiling. Of 410 differentially expressed genes, 286 were upregulated and 124 downregulated by mCD40L versus sCD40L. Gene ontology enrichment analysis revealed immune-stimulatory function as the most significant enriched biological process affected by upregulated transcripts, while those downregulated were critical for cell growth and division. Furthermore, immature dendritic cells (iDC) responded to mCD40L with enhanced maturation and activation over sCD40L evidenced by higher expression levels of CD83, CD86, HLA-DR and CD54, increased secretion of IL12 and IL10 and higher tumour-antigen (TA) uptake capacity. Furthermore, autologus CD3+ T cells responded to TA-loaded mCD40L-activated DC with increased proliferation and cytotoxic response (CD107a and IFN-γ-producing CD3+ CD8+ T cells) to the tumour-loaded autologous PBMCs compared to sCD40L. Thus, these data indicate that mCD40L enhances the immunostimulatory capacity over sCD40L. Furthermore, the ability of mCD40L to also directly induce cell death in CD40-expressing carcinomas, subsequently releasing tumour-specific antigens into the tumour microenvironment highlights the potential for mCD40L as a multi-faceted anti-cancer immunotherapeutic.

Heat-Killed Lactobacillus plantarum KCTC 13314BP Enhances Phagocytic Activity and Immunomodulatory Effects Via Activation of MAPK and STAT3 Pathways.

Identification of novel probiotic strains is of great interest in the field of functional foods. Specific strains of heat-killed bacteria have been reported to exert immunomodulatory effects. Herein, we investigated the immune-stimulatory function of heat-killed Lactobacillus plantarum KCTC 13314BP (LBP). Treatment with LBP significantly increased the production of TNF-α and IL-6 by macrophages. More importantly, LBP was able to enhance the phagocytic activity of macrophages against bacterial particles. Activation of p38, JNK, ERK, NF-κB, and STAT3 was involved in the immunomodulatory function of LBP. LBP treatment significantly increased production of TNF-α by bone marrow-derived macrophages and splenocytes, further confirming the immunostimulatory effect of LBP in primary immune cells. Interestingly, the immunomodulatory effects of LBP were much stronger than those of Lactobacillus rhamnosus GG, a well-known probiotic strain. These results indicate that LBP can be a promising immune-enhancing functional food agent.

Natural compound inducers of immunogenic cell death.

Accumulating evidence shows that the anti-cancer potential of the immune response that can be activated by modulation of the immunogenicity of dying cancer cells. This regulated cell death process is called immunogenic cell death (ICD) and constitutes a new innovating anti-cancer strategy with immune-modulatory potential thanks to the release of damage-associated molecular patterns (DAMPs). Some conventional clinically-used chemotherapeutic drugs, as well as preclinically-investigated compounds of natural origins such as anthracyclines, microtubule-destabilizing agents, cardiac glycosides or hypericin derivatives, possess such an immune-stimulatory function by triggering ICD. Here, we discuss the effects of ICD inducers on the release of DAMPs and the activation of corresponding signaling pathways triggering immune recognition. We will discuss potential strategies allowing to overcome resistance mechanisms associated with this treatment approach as well as co-treatment strategies to overcome the immunosuppressive microenvironment. We will highlight the potential role of metronomic immune modulation as well as targeted delivery of ICD-inducing compounds with nanoparticles or liposomal formulations to improving the immunogenicity of ICD inducers aiming at long-term clinical benefits.

Exosomes or Microvesicles, a Secreted Subcellular Organelle Contributing to Inflammation and Diabetes.

Type 1 and type 2 diabetes (T1D and T2D) are generally recognized as distinct disease entities based on the mechanism of induction of disease: T1D results from low or no insulin production due to the gradual loss of cells that produce insulin, whereas T2D is generally thought to occur as a result of the body’s development of resistance to insulin. Although the events initiating T1D and T2D might be very different, the progression to diabetes and complications are similar and involve interactions between the immune system and the metabolic system. Notably, chronic inflammation is a shared manifestation of the two types of diabetes (1,2). Recently, secreted microvesicles, particularly exosomes, were suggested to be intermediates linking inflammation to diabetes (3). The cellular origins of the vesicles and the genetic and environmental factors that cause an abnormal production of the vesicles are likely to be central in linking the inflammation to tissue-specific damage. For example, islet mesenchymal stem cells from autoimmune-prone animals were shown to release highly proinflammatory exosomes that contributed to T1D (4), and the low-grade inflammation in obesity may stimulate release of proinflammatory microvesicles and exosomes to accelerate T2D (5,6). “Extracellular vesicles” (EVs) is a collective term for different types of small-sized membrane vesicles or particles that are secreted by cells, including exosomes, microvesicles, apoptotic bodies, and virus-like particles. Figure 1 shows a generalization of their similarities and differences in biogenesis and immune-stimulatory function. There are many reasons that such organelles may be produced, including packaging of paracrine factors for intercellular communications, apoptosis resulting from breakdown of cells, and packaging of material for ingestion and disposal by scavenger cells (7). Viruses too have learned to take advantage of the vesicle biogenesis machinery to hide from immune surveillance to establish infections at immune-privileged sites …

Gr1-/low CD11b-/low MHCII+ myeloid cells boost T cell anti-tumor efficacy.

Conventional APCs that express MHC class II (MHCII) and co‐stimulatory molecules include dendritic cells (DCs) and macrophages. Beyond these conventional APCs, immune stimulatory cells have been more recently shown to extend to a class of atypical APCs, composed of mast cells, basophils, and eosinophils. Here, we describe a unique type of APC, Gr1−/lowCD11b−/low cells with a granularity and size characteristic of myeloid cells and with the ability to present Ag for crosspresentation. These cells constitutively express MHCII and the costimulatory molecules, CD80, CD86, and CD40. They do not express pan markers of myeloid DCs (CD11c), plasmacytoid DCs (Ly6C), or macrophages (F4/80), and their frequency is inversely correlated with myeloid‐derived suppressor cells (MDSCs) in tumor‐bearing mice. Among splenocytes, they are more abundant than DCs and macrophages, and they exhibit antitumor immune stimulatory function at a steady state without further activation, ex vivo. They are also found within the tumor bed where they retain their immune stimulatory function. Our findings suggest the use of these novel APCs in additional preclinical studies to further investigate their utility in APC‐based cancer immunotherapies.

Chitosan nanoparticle-based delivery of fused NKG2D-IL-21 gene suppresses colon cancer growth in mice.

Nanoparticles can be loaded with exogenous DNA for the potential expression of cytokines with immune-stimulatory function. NKG2D identifies major histocompatibility complex class I chain-related protein in human and retinoic acid early induced transcript-1 in mouse, which acts as tumor-associated antigens. Biologic agents based on interleukin 21 (IL-21) have displayed antitumor activities through lymphocyte activation. The NKG2D–IL-21 fusion protein theoretically identifies tumor cells through NKG2D moiety and activates T cells through IL-21 moiety. In this study, double-gene fragments that encode the extracellular domains of NKG2D and IL-21 genes were connected and then inserted into the pcDNA3.1(−) plasmid. PcDNA3.1–dsNKG2D–IL-21 plasmid nanoparticles based on chitosan were generated. Tumor cells pretransfected with dsNKG2D–IL-21 gene nanoparticles can activate natural killer (NK) and CD8+ T cells in vitro. Serum IL-21 levels were enhanced in mice intramuscularly injected with the gene nanoparticles. DsNKG2D–IL-21 gene nanoparticles accumulated in tumor tissues after being intravenously injected for ~4–24 h. Treatment of dsNKG2D–IL-21 gene nanoparticles also retarded tumor growth and elongated the life span of tumor-bearing mice by activating NK and T cells in vivo. Thus, the dsNKG2D–IL-21 gene nanoparticles exerted efficient antitumor activities and would be potentially used for tumor therapy.

MiR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.

GIFT4 fusokine converts leukemic B cells into immune helper cells.

BackgroundChronic lymphocytic leukemia (CLL) remains incurable with standard therapy, and is characterized by excessive expansion of monoclonal abnormal mature B cells and more regulatory immune properties of T cell compartment. Thus, developing novel strategies to enhance immune function merits further investigation as a possible therapy for CLL.MethodsWe generated a fusion cytokine (fusokine) arising from the combination of human GM-CSF and IL-4 (named GIFT4). Primary CLL cells were treated with GIFT4 or GM-CSG and IL-4 in vitro. GIFT4-triggered STAT5 signaling in CLL cells was examined by Western blot. The phenotype and secretome of GIFT4-treated CLL cells (GIFT4-CLL cells), and the immune stimulatory function of GIFT4-CLL cells on autologous T cells were analyzed by flow cytometry and luminex assay.ResultsGIFT4-CLL up-regulated the expression of co-stimulatory molecules CD40, CD80 and CD86 and adhesion molecule CD54. GIFT4-CLL cells secreted IL-1β, IL-6, ICAM-1 and substantial IL-2 relative to unstimulated CLL cells. GIFT4 treatment led to JAK1, JAK2 and JAK3-mediated hyper-phosphorylation of STAT5 in primary CLL cells, which is essential for GIFT4-triggered conversion of CLL cells. GIFT4-CLL cells directly propelled the expansion of autologous IFN-γ-producing CD314+ cytotoxic T cells in vitro, and that these could lyse autologous CLL cells. Furthermore, administration of GIFT4 protein promoted the expansion of human T cells in NOD-scid IL2Rγnull immune deficient mice adoptively pre-transferred with peripheral blood mononuclear cells from subjects with CLL.ConclusionGIFT4 has potent capability to converts primary CLL cells into APC-like immune helper cells that initiate a T cell driven anti-CLL immune response.

Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis

The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 μm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-β transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-β-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-β transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.

Role of Nucleotide-binding and Oligomerization Domain 2 Protein (NOD2) in the Development of Atherosclerosis.

NOD2 (nucleotide-binding and oligomerization domain 2) was initially reported as a susceptibility gene for Crohn's disease, with several studies focused on elucidating its molecular mechanism in the progression of Crohn's disease. We now know that NOD2 is an intracellular bacterial sensing receptor, and that MDP-mediated NOD2 activation drives inflammatory signaling. Various mutations in NOD2 have been reported, with NOD2 loss of function being associated with the development of Crohn's disease and other autoimmune diseases. These results suggest that NOD2 not only has an immune stimulatory function, but also an immune regulatory function. Atherosclerosis is a chronic inflammatory disease of the arterial wall; its pathologic progression is highly dependent on the immune balance. This immune balance is regulated by infiltrating monocytes and macrophages, both of which express NOD2. These findings indicate a potential role of NOD2 in atherosclerosis. The purpose of this review is to outline the known roles of NOD2 signaling in the pathogenesis of atherosclerosis.

MiRNA-181a is critical for the tolerogenic properties of plasmacytoid dendritic cells (TRAN3P.886)

Abstract Liver allografts are well tolerated and other solid organ allografts, when transplanted concurrently with livers, show improved graft outcomes. However, the mechanisms underlying “hepatic tolerance” have yet to be elucidated. Previous data show that hepatic dendritic cells (HDC) have diminished antigen presenting and immune stimulatory function compared with DC in lymphoid tissue. It has been hypothesized that immature plasmacytoid (p)DC are inherently tolerogenic with studies supporting this hypothesis. Our work confirms that HpDC prolong cardiac allograft survival (p<0.01) in a murine model compared to bulk and myeloid (m)DC. Micro-ribonucleic acid (miR) are short (15-22 nucleotides) RNA molecules which can post-transcriptionally regulate messenger RNA transcripts, resulting in translational repression or upregulation. We hypothesize that miR-181a, which we found is highly expressed in HpDC, plays a critical role in the tolerogenic properties of pDC. TLR 9 stimulation of pDC from miR-181a-/- mice exhibited higher levels of maturation markers and differential cytokine production as compared to wild-type C57BL/6 mice. More, there is altered phosphorylation of key members of signal transduction pathways, such as ERK1/2. Finally, in the same murine model of cardiac allograft transplantation, use of pDC from miR-181a-/- mice abrograted the tolerogenic properties seen with HpDC. Together, our data shows a critical role for miR-181a in the tolerance-inducing properties of pDC.

꽃송이버섯(Sparassis crispa) 추출물의 면역세포 활성화 및 항암 효과

꽃송이버섯(Sparassis crispa)은 항암 및 면역증강 효과가 뛰어나다고 알려져 있는 베타-글루칸의 함량이 건조중량의 40% 이상인 것으로 보고되고 있는 산림버섯이다. 일반적으로 버섯에서 추출한 베타-글루칸의 면역활성 및 항암 효과는 널리 알려져 있는 반면 꽃송이버섯 유래의 베타-글루칸에 의한 대식세포의 활성화 기작 및 자실체 추출물에 의한 항암 효과는 아직 보고되지 않았다. 본 연구에서는 꽃송이버섯 유래 베타-글루칸의 면역활성 효과를 확인하고자 마우스의 대식세포인 Raw 264.7 cell을 이용하였으며, 베타-글루칸 처리시 TNF-<TEX>${\alpha}$</TEX>와 interleukin-<TEX>$1{\beta}$</TEX>(IL-<TEX>$1{\beta}$</TEX>)와 같은 사이토카인의 발현 및 분비가 증가됨을 확인하였고, 이러한 과정에 필수적인 역할을 담당하는 전사 인자인 NF-<TEX>${\kappa}B$</TEX>가 활성화됨도 확인하였다. 뿐만 아니라 꽃송이버섯 추출물 처리 시 TNF-<TEX>${\alpha}$</TEX>의 분비가 증가됨과 함께 in vivo에서 암의 성장을 억제하는 기능이 있음을 확인하였다. 이러한 결과들은 꽃송이버섯이 면역활성을 증가시켜 암의 성장을 억제하는 건강 기능성 식품으로서의 개발 가능성이 높음을 의미하는 것이다. Sparassis crispa is an edible mushroom with medicinal properties that contains more than 40% <TEX>${\beta}$</TEX>-glucan. The role of S. crispa in regulating the functional activation of macrophages has yet to be fully elucidated. The objective of this study was to investigate the molecular mechanism underlying the immune-stimulatory function of S. crispa soluble <TEX>${\beta}$</TEX>-glucan and extracts on macrophages. In this study, we showed that S. crispa soluble <TEX>${\beta}$</TEX>-glucan was able to stimulate TNF-<TEX>${\alpha}$</TEX> and IL-<TEX>$1{\beta}$</TEX> production through NF-<TEX>${\kappa}B$</TEX> activation in Raw 264.7 cells. We also showed that S. crispa extracts could not only enhance TNF-<TEX>${\alpha}$</TEX> production in Raw 264.7 cells, but also suppress tumor growth in vivo. All of our results suggest that S. crispa could be developed as a promising immunostimulatory principle, applicable to cancer patients.

Silencing of the glucocorticoid-induced leucine zipper improves the immunogenicity of clinical-grade dendritic cells

BackgroundThe maturation cocktail composed of interleukin (IL)-6, IL-1β, tumor necrosis factor-α and prostaglandin E2 is considered the “gold standard” for inducing the maturation of dendritic cells (DCs) for use in cancer immunotherapy. Nevertheless, although this maturation cocktail induces increased expression of several activation markers, such as CD83, the co-stimulation molecules CD80, CD86 and CD40 and the chemokine receptor involved in DC homing in lymph nodes CCR7, the DC immune stimulatory function in vivo contrasts with this mature phenotype, and good clinical outcomes in patients with cancer treated with DC-based vaccines remain rare. MethodsPhenotypic characterization of the immunosuppressive status of DCs differentiated from peripheral blood mononuclear cells of healthy volunteers and matured with the “gold standard” cocktail was performed. Glucocorticoid-induced leucine zipper (GILZ) targeting small interfering RNA (siRNA) was electroporated into DCs after maturation to increase their immunogenicity. ResultsThe mature phenotype of DCs treated for 48 h with this cocktail was associated with the expression of several immunosuppressive regulators, including programmed cell death 1 ligand 1 (PD-L1), IL-10 and GILZ. Electroporation is a very efficient and safe way to deliver siRNA into DCs (80% of DCs receive at least one molecule of siRNA). Silencing GILZ in clinical-grade DCs by siRNA leads to a decrease of the PD-L1 expression associated with an increase in their IL-12 secretion and T-cell induction capability. ConclusionsGILZ silencing is a promising approach to achieving complete clinical-grade DC maturation and avoiding the immunosuppressive effects of the maturation cocktail on DCs intended for clinical use.