FasL Expression Research Articles

Carnosic acid enhances the anti-lung cancer effect of cisplatin by inhibiting myeloid-derived suppressor cells

Cisplatin and other platinum-based drugs are used frequently for treatment of lung cancer. However, their clinical performance are usually limited by drug resistance or toxic effects. Carnosic acid, a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), has been reported to have several pharmacological and biological activities. In the present study, the combination effect of cisplatin plus carnosic acid on mouse LLC (Lewis lung cancer) xenografts and possible underlying mechanism of action were examined. LLC-bearing mice were treated with intraperitoneal injection with cisplatin, oral gavage with carnosic acid, or combination with cisplatin and carnosic acid, respectively. Combination of carnosic acid and cisplatin yielded significantly better anti-growth and pro-apoptotic effects on LLC xenografts than drugs alone. Mechanistic study showed that carnosic acid treatment boosted the function of CD8+ T cells as evidenced by higher IFN-γ secretion and higher expression of FasL, perforin as well as granzyme B. In the meantime, the proportion of MDSC (myeloid-derived suppressor cells) in tumor tissues were reduced by carnosic acid treatment and the mRNA levels of iNOS2, Arg-1, and MMP9, which are the functional markers for MDSC, were reduced. In conclusion, our study proved that the functional suppression of MDSC by carnosic acid promoted the lethality of CD8+ T cells, which contributed to the enhancement of anti-lung cancer effect of cisplatin.

Insights into the Molecular Mechanism Accounting for Neutropenia in T-Large Granular Lymphocytes Leukemia

INTRODUCTION: Neutropenia represents the most frequent clinical manifestation in T-Large Granular Lymphocytes Leukemia (T-LGLL) patients. Literature data provided evidence of the involvement of soluble Fas Ligand (sFasL) in this process. Consistently, we confirmed that neutropenic T-LGLL patients were characterized by higher levels of sFasL than non neutropenic patients. We also demonstrated that FasL transcription was mediated by the Signal Transducer and Activator of Transcription (STAT)-3 and we showed that high STAT3 activation correlated with high levels of sFas. However, the mechanism through which STAT3 regulates FasL production still remains elusive.It is well known that FasL expression depends on post-transcriptional events involving ARE-binding proteins, such as Human antigen R (HuR). Important regulators of post-transcriptional modifications are microRNAs (miRNAs), that are small non-coding RNA molecules able to bind target mRNAs, promoting their degradation or blocking protein translation. Among them, miR-146b was identified to be induced by STAT3 in non-transformed cells.This work aims to determine whether miR-146b might regulate STAT3-mediated expression of FasL, thus playing a role in the pathogenesis of neutropenia in T-LGLL patients.METHODS: T-Large Granular Lymphocytes (T-LGLs) were purified by FACSAria cell sorter from PBMCs of untreated T-LGLL patients. High throughput and single miRNA analysis were carried out on purified LGLs by using the TaqMan® Human microRNA Array and Assays, respectively. Transfection with miR-146b mimic was performed using the Amaxa Nucleofactor and the Ingenio Electroporation Solution. Transcriptional and protein expression levels were evaluated by Real Time-PCR and Western Blot (WB) assays.RESULTS: By assessing the expression of 756 mature miRNAs on purified patients' T-LGLs, we identified miRNAs differentially expressed in patients characterized by neutropenia as compared to those with normal absolute neutrophil count (ANC); selected miRNAs were then analyzed for correlation with ANC. Among them, miR-146b expression was the only one correlated with ANC, being down-regulated in neutropenic patients. To investigate miR-146b role in neutropenia development, we transfected purified T-LGLs with a miR-146b mimic. We showed that restoration of miR-146b led to a decrease of FasL mRNA, without changes in the FasL primary transcript as compared to control, indicating that miR-146b affected FasL expression at a post-transcriptional level. However, FasL was not identified among the putative miR-146b target genes, suggesting that miR-146b could regulate FasL expression indirectly. Therefore, we checked for genes involved in mRNA stability and we found that the defective miR-146b expression lead to increased transcriptional levels of the mRNA stabilizer HuR, that is required for FasL expression in T-lymphocytes. Consistently, by WB assays, we demonstrated that in T-LGLs of neutropenic patients HuR endogenous protein levels were higher than in T-LGLs of non neutropenic ones. HuR-mediated FasL mRNA stabilization explained the increased FasL expression observed in neutropenic patients.In the end, we demonstrated the mechanism affecting miR-146b expression in the presence of STAT3 activation, pointing to a role of epigenetic modulation taking place, since the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) can restore STAT3-miR-146b axis.CONCLUSIONS: In this work we suggest a pathogenetic link between STAT3 activation, a defective miR-146b expression and neutropenia development in T-LGLL. Treatment with a demethylating agent may restore STAT3-dependent induction of miR-146b and may represent a new potential therapeutic strategy for the treatment of neutropenia in T-LGLL patients. DisclosuresNo relevant conflicts of interest to declare.

AT1Rknockdown confers cardioprotection against sepsis-induced myocardial injury by inhibiting the MAPK signaling pathway in rats.

Myocardial dysfunction is an important manifestation of sepsis. In addition, inactivation of the mitogen-activated protein kinase (MAPK)signaling pathway has been reported to be beneficial in sepsis. The current study used gene expression profiling to demonstrate the overexpression of angiotensin II type 1 receptor (AT1R) and activation of the MAPK signaling pathway in sepsis. In this study, we used a rat model of sepsis established by cecal ligation and puncture to explore the mechanism of AT1R silencing in relation to the MAPK signaling pathway on myocardial injury. Various parameters including blood pressure, heart rate, and cardiac function changes were observed. Enzyme-linked immunosorbent assay was used to measure the concentration of cardiac troponin T (TnT), cardiac troponin I (cTnI), and creatine kinase isoenzyme muscle/brain (CK-MB). Myocardial enzyme, tissue antioxidant capacity, mitochondria swelling, and membrane potential were also detected. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelingstaining was applied to measure cell apoptosis, andmessenger RNAand protein levels of apoptosis-related proteins (Fas ligand [Fasl], B-cell CLL/lymphoma [Bcl-2], p53) were also detected. Initially, sepsis rats exhibited decreased survival rate, but increased ejection fraction (EF), heart rate, and concentrations of TnT, cTnI, and CK-MB. Furthermore, decreased AT1R expression inactivated the MAPK signaling pathway (shown as decreased extracellular signal-regulated kinase and cyclic adenosine 3',5'-monophosphate response element binding protein expression), decreased EF, heart rate, and concentrations of TnT, cTnI, and CK-MB, but increased sepsis rat survival rate. Eventually, decreased AT1R expression inhibited myocardial cell apoptosis (shown as decreased apoptosis rate and p53 and Fasl expression as well as increased Bcl-2 expression). These findings indicated that AT1R silencing plays an inhibitory role in sepsis-induced myocardial injury by inhibiting the MAPK signaling pathway.

Ginger polysaccharides induced cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

In this study, ginger polysaccharide (GP) was obtained from ginger by enzymatic method, its chemical properties and antitumor activity were investigated. The results indicated that the composition and proportion of GP were l‑rhamnose, d‑arabinose, d‑mannose, d‑glucose and d‑galactose in a molar ratio of 3.64:5.37:3.04:61.03:26.91, GP had the characteristic absorption peak of polysaccharide. Congo red experiment showed that GP had a triple helix structure, which could have anti-tumor effect. Furthermore, MTT assay, cell morphology observation, nuclear morphology observation and reactive oxygen species observation demonstrated that GP had significant antitumor effect. Flow cytometry suggested that GP could promote apoptosis and arrest cells in G0-G1 phase. Real-time fluorescence quantification and Western blot revealed that GP could up-regulate the expression of Bax, Fas, FasL, caspase-3, p21 and p53, and down-regulate the expression of Bcl-2. These studies suggested that GP would be used as an antitumor drug in foods to promote the development of functional foods.

Apoptosis of tumor-infiltrating T lymphocytes: a new immune checkpoint mechanism.

Immunotherapy based on checkpoint inhibitors is providing substantial clinical benefit, but only to a minority of cancer patients. The current priority is to understand why the majority of patients fail to respond. Besides T-cell dysfunction, T-cell apoptosis was reported in several recent studies as a relevant mechanism of tumoral immune resistance. Several death receptors (Fas, DR3, DR4, DR5, TNFR1) can trigger apoptosis when activated by their respective ligands. In this review, we discuss the immunomodulatory role of the main death receptors and how these are shaping the tumor microenvironment, with a focus on Fas and its ligand. Fas-mediated apoptosis of T cells has long been known as a mechanism allowing the contraction of T-cell responses to prevent immunopathology, a phenomenon known as activation-induced cell death, which is triggered by induction of Fas ligand (FasL) expression on T cells themselves and qualifies as an immune checkpoint mechanism. Recent evidence indicates that other cells in the tumor microenvironment can express FasL and trigger apoptosis of tumor-infiltrating lymphocytes (TIL), including endothelial cells and myeloid-derived suppressor cells. The resulting disappearance of TIL prevents anti-tumor immunity and may in fact contribute to the absence of TIL that is typical of "cold" tumors that fail to respond to immunotherapy. Interfering with the Fas-FasL pathway in the tumor microenvironment has the potential to increase the efficacy of cancer immunotherapy.

Effect of sulfation and partial hydrolysis of polysaccharides from Polygonatum sibiricum on immune-enhancement

The aqueous polysaccharide from Polygonatum sibiricum was extracted and fractionated using anion-exchange chromatography to obtain F1 fraction. The F1 was chemically sulfated and partially acid-hydrolyzed for the production of its over-sulfated (OS1,2,3) and hydrolyzed (HP1,2,3) derivatives, in which the sulfate content of OS1,2,3 was 7.5–17.1%, and the Mw of HP1,2,3 ranged from 18.2 × 103 to 57.3 × 103 g/mol. Considerable RAW264.7 cell activation was observed by HP1,2,3 with NO production of 34.9, 44.3 and 42.7 μM, respectively, as well as the mRNA expression of cytokines (IL-1β, IL-6, IL-10 and IL-12). NK cell cytotoxicity against HT-29 cell was facilitated by OS1,2,3 treatment with the increased gene expressions of INF-γ, Granzyme-B, perforin, NKG2D, and FasL. RAW264.7 cells appeared to be activated via MR and TLR4 mediated signaling pathway, but CR3 and TRL2 might play a main role in stimulating NK cells. Overall, the present study suggests the potential application of polysaccharides from P. sibiricum in functional foods and pharmacological industries.

Fas/FasL pathway and cytokines in keratinocytes in atopic dermatitis - Manipulation by the electromagnetic field.

BackgroundAtopic dermatitis (AD) is one of the most frequent skin diseases. Changes of the keratinocytes functionality play a major role in the development of AD. For example, activation of the Fas (CD95)/FasL (CD178) pathway in AD does not lead to extensive apoptosis in skin. Binding of the Fas receptor to its protein ligand—FasL, which are present on the (AD)-modified keratinocytes, should result in the sequential induction of cell death, but there is no evidence of extensive apoptosis of these cells. This suggests that non-apoptotic mechanism of Fas/FasL pathway is commonly encountered, although not examined in the case of AD, phenomenon. An electromagnetic field, which was used to influence cultured cells in this study, can modulate proliferation, apoptosis, differentiation, and metabolism in various cells.ObjectiveHere, we evaluate the possibility to manipulate the immune activation of AD keratinocytes and their response to the electromagnetic field, which was not tested before.MethodsKeratinocytes isolated from the skin of healthy subjects (n = 20) and patients with atopic dermatitis (n = 20) as well as HaCaT and PCS-200-010 cell were exposed to the 900 MHz electromagnetic field for 60 minutes. Cytometric analysis of viability, Fas/FasL, p-ERK, p-p38 and p-JNK expression and Luminex analysis of cytokine concentration were performed in two-time points: 4 and 24 hours after the exposition.ResultsThis research has shown upregulated Fas, FasL, p-ERK, p-p38, and p-JNK expression along with increased cytokine secretion (IL-1β, IL-4, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-31 and TNFα) by keratinocytes derived from the skin of patients with the AD when compared with healthy control. Exposure of keratinocyte cultures obtained from AD patients to EMF resulted in a decrease of 1β, IL-4, IL-10, IL-12, I L-13, IL-17, IL-31 and TNFα levels. Keratinocytes derived from the skin of AD patients are characterized by elevated Fas and FasL expression when compared to healthy control.ConclusionApoptotic and nonapoptotic activation of the Fas/FasL-dependent signaling pathway may play a significant role in the pathogenesis of AD, by adjusting the local cytokine and chemokine environment at the site of inflammation. Moreover, the electromagnetic field exhibits strong immunomodulatory effects on AD-modified keratinocytes.

THE STUDY OF IMMUNOHISTOCHEMICAL EXPRESSION OF CERTAIN PRO- AND ANTIAPOPTOTHIC FACTORS IN ENDOMETRY OF PATIENTS WITH ADENOMIOSIS

Objective: to study the immunohistochemical expression of proand anti-apoptotic factors — FasL(CD95), TNF-a, p65 NFkB, and granzyme B in the endometrium of patients with adenomyosis. Material and Methods. The endometrial samples, taken during the proliferative phase of the cycle, while carrying out paypel biopsy and hysteroscopy in 41 women aged 37 to 48 years with adenomyosis. The comparison group consisted of 25 women with infertility. The study of TNF-a, Granzym B, NFkB immunohistochemical expression in the development and progression of adenomyosis was performed using monoclonal antibodies. The results of reaction were evaluated by semiquantitative method. Statistical analysis was performed using the software package Biostat Apps MicrosoftR Excel full-featured office Microsoft Office 2010 package. Results. In the comparison group, the expression of TNF-a and NFkB in endometrium was not evident. In the group of patients with adenomyosis, a marked expression of these markers in epithelial and stromal cells was observed. Granzym B expression was detected in the cytoplasm of leucocytes of the stroma of the endometrial functional layer with no statistically significant differences between the groups. Conclusion. Presumably, given the expression peaks of both TNF-a, p65 NFkB, the observed in adenomyosis increase (compared to normal) in expression of these molecules does not mean readiness of epithelial cells of the endometrial functional layer to apoptosis, as is the norm, upon the occurrence of menstruation, but it means readiness of these cells to proliferate. The absence of statistically significant differences in the number of CD56-positive endometrial stromal granulocytes and endometrial NK-cells, determined by Granzym B expression in these cells, is also an indirect evidence of a low level of readiness to apoptosis. And statistically significantly less expression of FasL(CD95) in women with adenomyosis. That is, TNF-a and NFkB at adenomyosis, probably, play the role of cell survival factors.

Characteristics of peripheral V\u03b32V\u03b42 T cells in interferon-\u03b3 release assay negative pulmonary tuberculosis patients

BackgroundIt is not fully explained why some active tuberculosis patients show negative interferon-γ release assays (IGRAs). In this study, we tried to explore associations of IGRAs with the characteristics of peripheral Vγ2Vδ2 T cells and their functions of producing cytokines.Methods32 pulmonary tuberculosis patients were enrolled and divided into two groups according to their IGRAs results: 16 with IGRA-negative as test group and 16 with IGRA-positive as control group. Chest X-rays and T-SPOT.TB tests were performed and the severity of the lung lesions was scored. The amount of Vγ2Vδ2T cell and their expression levels of the apoptosis-related membrane surface molecule Fas and FasL in peripheral blood were analyzed by flow cytometry, and the function of secreting cytokines (IFN-γ, TNF-α and IL-17A) of Vγ2Vδ2 T cell were determined by intracellular cytokine staining.ResultsThe IGRA-negative TB patients had more lesion severity scores and displayed reduced peripheral blood Vγ2Vδ2 T cell counts (p = 0.009) as well as higher Fas and FasL expression in peripheral blood Vγ2Vδ2 T cells (p = 0.043, 0.026). A high lesion severity score was correlated with a decreased Vδ2+ T cell number and increased Vγ2Vδ2 T cells Fas/FasL expression leve in the peripheral blood (p = 0.00, P < 0.01). The function of secreting cytokines was slightly impaired in IGRA-negative TB patients (p = 0.402). There is no significant differences in expression levels of Fas and FasL in CD4+ T cells (p = 0.224, 0.287) or CD8+ T cells (p = 0.184, 0.067) between test and control groups.ConclusionCompared with IGRA-positive TB patients, the IGRA-negative TB patients had more lesion severity scores, the number of Vγ2Vδ2 T cells decreased and the function of secreting cytokines impaired. In addition, we suggest that increased expression of Fas/FasL triggers Vγ2Vδ2 T cell apoptosis.

Insight into the negative impact of ionic liquid: A cytotoxicity mechanism of 1-methyl-3-octylimidazolium bromide

Ionic liquids (ILs) as a green replacement for volatile organic solvents are increasingly used in large-scale commercial applications. A good understanding of the toxic mechanisms and environmental impact of ILs is neede to reduce the risk for human health and the environment. For this purpose, we aimed to evaluate the possible impacts of 1-methyl-3-octylimidazolium bromide ([C8mim]Br) exposure on human hepatocellular carcinoma (HepG2) cells as to elucidate the cytotoxic mechanism of [C8mim]Br. Biochemical assays revealed that [C8mim]Br exposure altered the protein levels of heat shock protein 70 (HSP70) and HSP90, generally inhibiting total antioxidative capacity (T-AOC), depleting heme oxygenase-1 (HO-1) and increasing transcription and activity of inducible nitric oxide synthase (iNOS) in HepG2 cells. These results indicated that [C8mim]Br may induce biochemical disturbances and cause oxidative stress in HepG2 cells. Moreover, increased phosphorylation of p53, mitochondrial membrane disruption, cyclooxygenase-2 activation, Bcl-2 family protein modulation, cytochrome c and Smac/DIABLO release, and inhibition of apoptosis inhibitory protein-2 (c-IAP2) and survivin were also observed in [C8mim]Br-treated cells, suggesting that [C8mim]Br-induced apoptosis might be mediated by the mitochondrial pathway. Further research showed that [C8mim]Br exposure increased tumour necrosis factor α (TNF-α) transcription and content and promoted the expression of Fas and FasL, indicating that TNF-α and Fas/FasL are involved in the apoptosis induced by [C8mim]Br. Additionally, [C8mim]Br cytotoxicity was partly inhibited by N-acetyl-cysteine (NAC), and NAC reversed [C8mim]Br-mediated mitochondrial dysfunction and blocked apoptotic events by inhibiting the generation of reactive oxygen species (ROS). This work first demonstrated that the ROS-mediated mitochondrial and death receptor-initiated apoptotic pathway is involved in [C8mim]Br-induced HepG2 cell apoptosis.

Effects of fetal umbilical cord blood mesenchymal stem cells on γδT lymphocyte function and its related mechanism

Objective To explore the effects of fetal umbilical cord blood mesenchymal stem cells on γδT lymphocyte function and its related mechanism. Methods The Human peripheral blood mononuclear cells (PBMCs) or γδT cells were activated and expanded with 2 μg/ml pamidronate and 100 IU/ml interleukin-2 (IL-2) with or without the presence Fetal umbilical cord blood mesenchymal stem cells (UCMSCs). Flow cytometry was performed to evaluate the effects of UC-MSCs on proliferation, cytokine expression and cytotoxicity of γδT cells; besides, flow cytometry was used to detect the effects of UC-MSCs on Fas-L and TRAIL expression in γδT cells and γδT cell apoptosis as well. Results UC-MSCs inhibited γδT cell proliferation in a dose-dependent but cell-contact independent manner; when PBMCs and UC-MSCs were cultured at a ratio of 3∶1 and 40∶1, IFN-γ+ cells decreased by 46.7% and 31.4%, respectively (P=0.001, P=0.021), granzyme B positive cells increased by 79.7%, 165%, respectively, while the ratio of TNF-α and IL-10 positive cells did not change significantly compared with the control group (P=0.062); Cytotoxicity test results showed that, UCMSCs inhibited the cytotoxicity ofγδT cells against influenza virus H1N1 infected A549 cells, and when PBMCs and UC-MSCs were cultured at a ratio of 3∶1 and 40∶1, Fas-L+ γδT cells decreased by 87.6% and 37.5% (P=0.000, 0.009), the proportion of TRIAL+ γδT cells decreased by 43.2% and 18.7% (P=0.037, 0.042), however, UC-MSCs did not affect γδ T cell apoptosis (P=0.070, 0.064). Conclusion UC-MSCs can suppress γδT cells proliferation and cytotoxicity and modulated their cytokine production, and Fas-L and TRAIL were involved in the regulation. Key words: Umbilical cord blood mesenchymal stem cells; γ δ T cells; Proliferation; Cytokines; Apoptosis

NFATc1 is a tumor suppressor in hepatocellular carcinoma and induces tumor cell apoptosis by activating the FasL-mediated extrinsic signaling pathway.

Nuclear factor of activated T cells (NFAT) is a family of transcription factors that have important functions in many tumors. However, the expression level and functional role of NFAT in hepatocellular carcinoma (HCC) remain unclear. In this study, we showed that NFATc1 expression was decreased in both HCC tissues and cell lines. Low expression of NFATc1 was correlated with larger tumor size, advanced tumor‐node‐metastasis (TNM) stage, high serum AFP level, and liver cirrhosis. Furthermore, patients with low NFATc1 expression exhibited poor prognosis. Ectopic expression of NFATc1 in HCC cells inhibited proliferation and colony formation, leading to G1 arrest and induction of apoptosis. In addition, we demonstrated that NFATc1 increased Fas ligand (FasL) expression by directly binding to its promoter and activated the extrinsic apoptotic pathway. NFATc1 and FasL expression patterns and their prognostic value for patients with HCC were also evaluated in TCGA Liver Hepatocellular Carcinoma database. Knock‐down of FasL expression by siRNA in HCC cell lines abolished NFATc1's antiproliferative and pro‐apoptotic effects. In conclusion, NFATc1 is frequently inactivated in HCC and functions as a tumor suppressor in liver carcinogenesis. Ectopic expression of NFATc1 in HCC cells induces apoptosis by activating the FasL‐mediated extrinsic signaling pathway.

Notch signaling pathway suppresses CD8+ T cells activity in patients with lung adenocarcinoma

Evolution and progression of cancer always leads to CD8+ T cells dysfunction/exhaustion. Controversy remains as to the role of Notch signaling pathway in CD8+ T cells regulation in tumorigenesis. Thus, the aim of this study was to investigate the immunomodulatory activity of Notch signaling pathway to peripheral and lung-resident CD8+ T cells in patients with lung adenocarcinoma. Forty-eight lung adenocarcinoma patients and twenty healthy individuals were enrolled in the current study, and CD8+ T cells were purified from both peripheral bloods and bronchoalveolar lavage fluids. Notch receptor mRNA expression was semi-quantified by real-time PCR. Cytolytic and noncytolytic activity of CD8+ T cells evaluated in direct and indirect contact co-culture with A549 cells in response to Notch signaling inhibition by measuring of lactate dehydrogenase release and cytokines production. Expression of Fas ligand (FasL), perforin, and granzyme B were also assessed by flow cytometry. Notch2 mRNA expression was elevated in both peripheral and lung-resident CD8+ T cells in lung adenocarcinoma patients, however, did not correlated with tumor stages or epidermal growth factor receptor mutation. Peripheral CD8+ T cells from healthy individuals exhibited stronger cytotoxicity in direct contact co-culture system, which was not influenced by Notch signaling inhibition. Moreover, suppression of Notch signaling augmented cytotoxicity of peripheral and lung-resident CD8+ T cells from lung adenocarcinoma patients in direct contact co-culture system, and promoted interferon-γ production in both systems. This process was accompanied by increased expression of FasL and perforin within CD8+ T cells. The current data revealed a potential immunosuppressive property of Notch signaling pathway to CD8+ T cells probably via inhibition of FasL and perforin in lung adenocarcinoma patients.

RETRACTED ARTICLE: A novel Fas-binding outer membrane protein and lipopolysaccharide of Leptospira interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway

Leptospira interrogans is the major causative agent of leptospirosis, an emerging, globally spreading zoonotic infectious disease. The pathogen induces macrophage apoptosis, but the molecular basis and mechanism remain unknown. In the present study, we found that L. interrogans caused apoptosis of phagocytosis-inhibited macrophages, and the product of the L. interrogans LB047 gene (Lep-OMP047) was the unique protein captured by mouse and human Fas proteins. The recombinant expressed Lep-OMP047 (rLep-OMP047) strongly bound mouse and human Fas proteins with equilibrium association constant (KD) values of 5.20 × 10−6 to 2.84 × 10−9 M according to surface plasmon resonance measurement and isothermal titration calorimetry. Flow-cytometric examination showed that 5 μg rLep-OMP047 or 1 μg lipopolysaccharide of L. interrogans (Lep-LPS) caused 43.70% or 21.90% early apoptosis in mouse J774A.1 macrophages and 28.41% or 15.80% for PMA-differentiated human THP-1 macrophages, respectively, but the apoptosis was blocked by Fas-antagonizing IgGs, Fas siRNAs, and caspase-8/-3 inhibitors. Moreover, Lep-OMP047 was significantly upregulated during infection of macrophages. Lep-LPS promoted the expression and cytomembrane translocation of Fas and FasL in macrophages. The JNK and p38 MAPK but not ERK signaling pathways, as well as the transcription factors c-Jun and ATF2 but not CHOP, mediated Lep-LPS-induced Fas/FasL expression and translocation. TLR2 but not TLR4 mediated Lep-LPS-induced JNK/p38 MAPK activation. Therefore, we demonstrated that a novel Fas-binding OMP and LPS of L. interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway.

β-Bourbonene attenuates proliferation and induces apoptosis of prostate cancer cells.

Sesquiterpenes have antitumor, anti-inflammation, and anti-fungal effects. β-bourbonene is a kind of sesquiterpene, but its pharmacological effect has not been studied. The present study was conducted in order to investigate the potential anticancer effects of β-bourbonene on human prostate cancer PC-3M cells. PC-3M cells were incubated with 0, 25, 50, 100 µg/ml of β-bourbonene. Cell Counting Kit-8 (CCK-8) detection showed that compared with the control group, β-bourbonene inhibited the growth of PC-3M cells in a dose-dependent manner. G0/G1 phase arrest was observed by β-bourbonene by using flow cytometry. TUNEL staining and Annexin V/PI dual-staining method revealed that apoptosis was found in cells with β-bourbonene treatment, and the quantity of apoptotic cells was increased with the elevation in concentration. The mRNA and protein expression levels of Fas and FasL in the drug-treatment group were significantly elevated. Furthermore, the western blot assay also indicated that with an increase in the concentration of β-bourbonene, the protein expression of Bax in the drug-treatment group was significantly elevated, while a decrease was identified in the protein expression of Bcl-2. Taken together, β-bourbonene can inhibit the proliferation and simultaneously, induce apoptosis and G0/G1 arrest of prostate cancer PC-3M cells, which may be realized by upregulation of mRNA expression of Fas and FasL, increase of Bax protein expression and decrease of Bcl-2 protein expression.

Increased killer B cells in chronic HCV infection may lead to autoimmunity and increased viral load

Regulatory B (Breg ) cells are characterized by various membrane markers and the secretion of different inhibitory cytokines. A new subset of Breg cells was identified as CD5hi Fas-ligand (FasL)hi . Their main reported role is to suppress anti-viral and anti-tumour immune responses, and, hence they have been dubbed 'killer' B cells. In this study, we aim to assess the role of these cells in chronic hepatitis C virus (HCV) infection, and determine if they contribute to the increased viral load and persistence of HCV and its related autoimmunity. (i) FasL expression on CD5hi B cells is increased significantly in HCV-infected patients compared to healthy individuals [28·06±6·71 mean fluorescence intensity (MFI)±standard error of the mean (s.e.m.), median = 27·9 versus 10·87±3·97 MFI±s.e.m., median = 10·3, respectively, P<0·0001]. (ii) Killer B cells from HCV patients increased autologous CD4+ T cell apoptosis compared to the apoptosis in healthy individuals [39·17%±7·18% mean±standard deviation (s.d.), median = 39·6 versus 25·92±8·65%, mean±s.d., median = 24·1%, P<0·0001, respectively]. A similar increase was observed in CD8+ T cell apoptosis (54·67±15·49% mean±s.d., median=57·3 versus 21·07%±7·4%, mean±s.d., median=20%, P=0·0006, respectively). (iii) By neutralizing FasL with monoclonal anti-FasL antibodies, we have shown that the induction of apoptosis by killer B cells is FasL-dependent. (iv) Increased expression of FasL on CD5hi B cells is correlated positively with an increased viral load and the presence of anti-nuclear antibodies and rheumatoid factor in HCV. This is the first study in which killer B cells have been suggested to play a pathogenic role in HCV. They seem to be involved in HCV's ability to escape efficient immune responses.

Nuclear factor-kappa B activation inhibits proliferation and promotes apoptosis of vascular smooth muscle cells

To investigate the role of nuclear factor-kappa B (NF-κB) performed in cell proliferation and apoptosis of vascular smooth muscle cells (VSMCs), and to assess the mechanisms. Human aorta VSMCs were divided into control, NF-κB inhibitor, NF-κB overexpression + NF-κB inhibitor, control vector + NF-κB inhibitor, NF-κB overexpression, and control vector groups. NF-κB overexpression vector was constructed and transfected into VSMCs. Proliferation of VSMCs in each group was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide. Apoptosis of VSMCs was detected by flow cytometry. The expression of NF-κB, FasL, and hypertension-related gene (HRG-1) was measured by Western blotting. NF-κB overexpression vector was constructed correctly by restriction endonuclease, and the results showed that the activation of NF-κB could inhibit the proliferation of VSMCs. The results of flow cytometry also confirmed that NF-κB overexpression promoted apoptosis of VSMCs. Mechanically, NF-κB overexpression could up-regulate the expression of FasL and HRG-1. NF-κB overexpression promotes apoptosis and inhibits cell proliferation of VSMCs. The mechanisms might be regulated by promoting FasL and HRG-1 expression.

Abstract 4648: Using the IntelliCyt iQue Screener to simultaneously profile antibody dependent cell cytotoxicity and cytokine release of therapeutic antibodies

Abstract Therapeutic antibodies can mediate cancer cell killing through a variety of mechanisms including antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent killing (CDC). ADCC is induced through the binding of the antibody Fc region to Fc receptors (FcRs) expressed on effector cells of the immune system. Granule exocytosis of perforin, granzyme B, the induction of Fas ligand (FasL) expression on immune cells and the release of cytokines are major mechanisms involved in targeted cancer cell killing. Traditional approaches to screen for antibody Fc effector functions focus on ADCC using a homogenous live/dead readout, which greatly limits the contextual and correlative value of the screening data. To overcome this limitation, a multiplex screening assay profiling ADCC using multiple cell death endpoints, and quantitating secreted proteins /cytokines was developed and analyzed using Intellicyt's iQue Screener PLUS and integrated ForeCyt software. The iQue Screener Plus is a high throughput flow cytometry platform featuring 3 lasers with 13 fluorescent channels and can sample a 384-well plate in 20 minutes. As a proof of concept, a small set of therapeutic antibodies directed against the same tumor antigen were used to induce immune cell-mediated killing of tumor cells using different effector to target cell ratios and a range of antibody concentrations. To demonstrate antibody specificity, antigen positive target cells, negative control target cells and effector immune cells were barcoded with different encoding dyes and included in the same well. At specific times, a small aliquot from each well was transferred to a new assay plate to run a multiplexed cell/bead mixture assay by the addition of a cocktail of fluorescent immunophenotyping antibodies, fluorescent dyes measuring unique apoptosis parameters, and a panel of QBeads for secreted protein detection. The samples were assayed on the IntelliCyt iQue Screener PLUS with simultaneous data analysis including multi-plate analysis for cell killing kinetics. Positive/negative target cells and immune cells were digitally segregated by differently encoded fluorescence. Time-dependent cell killing of antigen positive target cells were observed using the apoptosis markers mitochondria depolarization staining and cell membrane integrity, whereas little killing was measured in antigen negative cells. The antibody set showed a range of ADCC mediated killing and granule exocytosis/ cytokine release suggesting differences in Fc effector functions. These results highlight the streamlined workflow on IntelliCyt iQue Screener PLUS platform to profile therapeutic antibodies in a functional ADCC/cytokine release assay predicting their clinical efficacy. Citation Format: John O'Rourke. Using the IntelliCyt iQue Screener to simultaneously profile antibody dependent cell cytotoxicity and cytokine release of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4648.

Immunoprotective responses against murine sarcocystosis by β - Irradiated sporocysts

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using β-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 μSv β-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4+ and CD8+ T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8+ T, NK1.1+ and FasL+ cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1+ & FasL+ cells, serum IFN-γ and intestinal IgA, while CD4+ and CD8+ T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. β-irradiated sporocysts with 1.84 μSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver.

Glycogen synthase kinase 3 inhibition lowers PD-1 expression, promotes long-term survival and memory generation in antigen-specific CAR-T cells

Successful remission in hematological cancers by CAR-T cell immunotherapy has yet to be replicated in solid tumors like GBM. A significant impediment of CAR-T immunotherapy in solid tumors is poor exposure of T cells to tumor antigens resulting in suboptimal CAR-T cell activation, which ultimately fails to induce a robust anti-tumor immune response. Costimulatory moieties in advanced-generation CARs, along with additional IL2 therapy has been shown to be insufficient to overcome this hurdle and have its cytotoxic limitations. GSK3 is constitutively active in naïve T cells and is transiently inactivated during T cell activation resulting in rapid T cell proliferation. Pharmacologic inhibition of GSK3 in GBM-specific CAR-T cells reduced FasL expression, increased T cell proliferation and reduced exhaustion by lowering PD-1 levels resulting in the development of CAR-T effector memory phenotype. Treatment with GSK3-inhibited CAR-T cells resulted in 100% tumor elimination during the tumor-rechallenge experiment in GBM-bearing animals and increased accumulation of memory CAR-T cells in secondary lymphoid organs. These adjuvant-like effects of GSK3 inhibition on activated CAR-T cells may be a valuable adjunct to a successful implementation of CAR-T immunotherapy against GBM and other solid tumors.