FasL Expression Research Articles

Heat stress decreases egg production of laying hens by inducing apoptosis of follicular cells via activating the FasL/Fas and TNF-α systems

Heat stress (HS) causes significant economic losses in the poultry industry every year. However, the mechanisms for the adverse effects of HS on avian follicular development are largely unknown. The aim of this study was to test whether HS induces apoptosis of follicular cells and impairs egg production by activating the FasL/Fas and tumor necrosis factor (TNF)-α systems. To this end, Hy-Line Brown laying hens, at 32 wk of age, were either exposed to HS of 35°C to 37°C or maintained at 24°C to 26°C (control) for 5 D. At the end of the HS period, follicle numbers, apoptosis, FasL/Fas and TNF-α activation, oxidative stress, and hormone secretion were examined in ovarian follicles. Egg production was observed daily during both the stressed (day S1–S5) and the poststress recovery (day R1–R15) periods. The results demonstrated that HS on hens significantly 1) decreased laying rates from day S3 to R6; 2) reduced numbers of large yellow and hierarchical follicles; 3) triggered apoptosis while increasing the expression of FasL, Fas, TNF-α, and TNF-receptor 1 in small and large yellow follicles; and 4) increased levels of oxidative stress, corticotrophin-releasing hormone, and corticosterone while decreasing the estradiol/progesterone ratio in follicular fluid in small and large yellow follicles. Taken together, the results suggested that hen HS impaired egg production by reducing the number of follicles through inducing apoptosis and that it triggered apoptosis in follicular cells by activating the FasL/Fas and TNF-α systems.

The therapeutic effect of Interleukin-18 on hypertrophic scar through inducing Fas ligand expression

ObjectiveAmong downstream interleukin-18 (IL-18) targets, Fas ligand (FasL) in particular, has been strongly implicated in many conditions. Our study aims to explore the role of IL-18 in hypertrophic scar through enhancing FasL expression. MethodsIL-18 expression in hypertrophic scar tissues and normal tissues were explored by immunohistochemistry, qRT-PCR and Western blotting, and the expression of IL-18 in normal skin fibroblasts and hypertrophic scar fibroblasts by immunofluorescence. Hypertrophic scar fibroblasts treated with recombinant human IL-18 (rhIL-18) were assessed with MTT, Annexin V-FITC/PI, qRT-PCR, ELISA and western blotting. In the hypertrophic scar of rabbit ears, rhIL-18 was injected to determine histological changes with HE and Masson staining. Additionally, the scars were rated based on contour and overall severity using a visual analog scale scores (VAS). ResultsIL-18 was decreased in hypertrophic scar tissues and fibroblasts compared to normal skin tissues and fibroblasts, respectively. Decreased proliferation and increased apoptosis of hypertrophic scar fibroblasts were found after rhIL-18 treatment with enhanced expression of FasL, sFasL FADD, Caspase-8, Caspase-9 and Caspase-3 in a dose-dependent manner. The VAS and thickness of scars in rabbit ears was decreased as time went on after rhIL-18 treatment, with decreases in scar elevation index (SEI) and the increases in FasL expression. ConclusionIL-18 curbs proliferation and promotes apoptosis of hypertrophic scar fibroblasts by enhancing FasL expression. IL-18is a potential target for treatment of hypertrophic scar.

Cardiac apoptosis caused by elevated cholesterol level in experimental autoimmune myocarditis

It has been reported that cholesterol-lowing agents can ameliorate severity of myocarditis. However, the beneficial effect of the agents has been claimed to be independent of cholesterol reduction as there is no significant change in the plasma cholesterol level in myocarditis. In the present study, using experimental autoimmune myocarditis (EAM) rats as an animal model, we demonstrated that EAM induced elevation of cholesterol level and impaired cholesterol efflux capacity in the cardiac tissue. Moreover, serum high-density lipoprotein (HDL) content was reduced and HDL function associated protein Paraoxonase 1 (PON1) activity was decreased. Besides, the major structural protein within HDL, Apolipoprotein A1 (ApoA1) expression in the cardiac tissues was significantly reduced while the level of serum ApoA1 was not significantly altered. Importantly, cholesterol depleting agent methyl-β-cyclodextrin (MβCD) alleviated the development of EAM, as monitored by decreased ratio of heart weight to body weight (HW/BW), decreased infiltration of inflammatory cells and collagen deposition, improved cardiac function, reduced expression of apoptosis-related protein Bax, Fas, FasL and caspase-3 and increased level of anti-apoptotic protein Bcl-2. These results suggest that reduction of cholesterol level in cardiac tissue could suppress EAM-induced cardiac apoptosis through both intrinsic and extrinsic apoptotic pathways.

Effect of Glucose Metabolism-Associated Protein-1 on Exendin-4-Treated Pancreatic Islet Cell Function

Exendin-4 regulates blood sugar. Glucose metabolism-associated protein-1 (GMRP-1) regulates islet cell function. Therefore, this paper intends to analyze the effect of GMRP-1 on Exendin-4treated islet cells. The islet cell MIN6 cells were separated into control group, Exendin-4 group, GMRP-1 overexpressing+ Exendin-4 group, and GMRP-1 siRNA+ Exendin-4 group followed by analysis of the expression of GMRP-1 by quantitative real-time PCR and Western blot, cell proliferation by MTT, cell apoptosis by flow cytometry, Bcl-2, Bax, Fas and Fas-L expression by real-time PCR, as well as caspase-3 activity, reactive oxygen species (ROS) and superoxide dismutase (SOD) activity. Exendin-4 and GMRP-1 overexpression+ Exendin-4 significantly increased GMRP-1 expression and promoted cell proliferation, decreased apoptotic rate and Caspase-3 activity, increased Bcl-2 and Fas-L expression, reduced Fas and Bax expression, as well as decreased ROS generation and increased SOD activity (P < 0 05), and the changes in GMRP-1 overexpression+ Exendin-4 group was more significant (P < 0 01). However, GMRP-1 siRNA+ Exendin-4 group significantly down-regulated GMRP-1 expression, decreased MIN6 cell prolifer- ation, increased cell apoptosis and Caspase-3 activity, decreased Bcl-2 and Fas-L expression, elevated Fas and Bax expression, increased ROS content and decreased SOD activity compared to control group and Exendin-4 group (P < 0 05). Up-regulation of GMRP-1 expression promoted Exendin-4-induced proliferation and inhibited apoptosis of islet cells. Down-regulation of GMRP-1 expression reversed Exendin-4's effect on islet cells.

LncRNA TUG1 promotes endometrial fibrosis and inflammation by sponging miR-590-5p to regulate Fasl in intrauterine adhesions

Intrauterine adhesion (IUA) is one of the most common reproductive system diseases in women worldwide. The role of lncRNAs in multiple diseases has been confirmed, but the role and mechanism of lncRNA taurine upregulated gene 1 (TUG1) in the progression of IUA need to be elucidated further. Here, we found that lncRNA TUG1 was upregulated in the endometrial tissues of IUA and TGF-β1-treated human embryonic stem cells (hESCs). Moreover, lncRNA TUG1-silenced alleviated TGF-β1-induced the proliferation and migration abilities of hESCs and enhanced inflammatory cytokines secretion in vitro. In vivo experiments showed that inhibition of lncRNA TUG1 promoted endometrium regeneration in IUA rats through downregulating inflammatory response and epithelial-to-mesenchymal transition (EMT) process. Mechanistically, lncRNA TUG1 suppression attenuated EMT process and inflammation through competitively binding miR-590-5p to downregulate Fasl expression. Collectively, our findings provide vital theoretical evidence for explaining the mechanisms of the lncRNA TUG1/miR-590-5p/Fasl axis in the progression of IUA, and may provide a new biomarker for the treatment of IUA patients.

Neuroprotective effect of Umbelliferone against Cerebral ischemia/Reperfusion induced neurological deficits: in-vivo and in-silico studies

Inflammatory pathway is the significant marker of neuro-inflammation and plays a significant role in the expansion of cerebral ischemia/reperfusion injury. Umbelliferone (UF), 7-hydroxy coumarin, has been already proved for its anti-inflammatory and anti-oxidative effects against ischemic brain injury in the rodent model, but its underlying pharmacological mechanism for neuro-protection remain unclear. In this study, we try to explore the neuro-protective effect of umbelliferone against ischemia/Reperfusion induced neurological deficits in rats and explore the underlying mechanism. Inserting thread into the middle cerebral artery was used to induce the ischemic stroke model. The rats were treated with the umbelliferone (5, 10 and 20 mg/kg) for 14 days prior to the ischemic stroke. At the end of the experimental study, brain infarction volume, neurological score, brain edema, pro-inflammatory cytokines, inflammatory mediator were estimated in the region of brain and serum. The mRNA expression of Toll-like receptor-4 (TLR4), myeloid differentiation factor 88 (MyD88), Fas and FasL were also estimated at the end of the study. Dose dependently treatment of umbelliferone down-regulated the neurological score, brain infarction, inflammatory mediator (TNF-α, IL-1β, IL-6, COX-2, NF-kB and PGE2) in the serum and brain tissue as compared to I/R induced control group rats. Umbelliferone also reduced the expression of TRL4, MyD88, Fas and FasL as compared to I/R control group rats. Umbelliferone also decreased the level of nuclear factor kappa B (NF-kB) compared to MACO control group rats. Collectively, the obtained result showed that the umbelliferone protected the brain against the ischemic injury in the rats through the inhibition of inflammatory pathway. Communicated by Ramaswamy H. Sarma

Daphnetin induces apoptosis in fibroblast-like synoviocytes from collagen-induced arthritic rats mainly via the mitochondrial pathway

Rheumatoid arthritis (RA) is a chronic, symmetric, systemic autoimmune disease. Because insufficient apoptosis of fibroblast-like synoviocytes (FLS) is an important characteristic of RA, promoting apoptosis is considered a potential therapeutic tool for treating RA. We have previously found that daphnetin (7,8-dihydroxycoumarin, DAP) has a pro-apoptotic effect on fibroblast-like synoviocytes from collagen-induced arthritis (CIA) rats. In the present study, we further investigated the mechanisms of DAP-induced apoptosis in CIA-FLS. CIA-FLS were incubated with DAP for 48 h in the presence or absence of caspase inhibitors, including inhibitors of caspase-3, caspase-8, or caspase-9 or a pan-caspase inhibitor; then, a series of experiments were performed to evaluate the mechanisms of DAP-induced apoptosis. Our results showed that DAP markedly decreased cell viability and induced the apoptosis of CIA-FLS along with typical morphological and ultrastructural changes; moreover, DAP increased FasL, cytochrome c (Cyt-c), Bax, caspase-3, caspase-8, and caspase-9 mRNA expression and Bax, caspase-3, caspase-8, and caspase-9 protein expression. In contrast, DAP decreased Bcl-2 mRNA and protein expression and promoted the release of Cyt-c from the mitochondria into the cytosol; these effects were attenuated to varying degrees by pre-treatment with caspase inhibitors, especially with caspase-3 or caspase-9 inhibitors or a pan-caspase inhibitor. In conclusion, the current findings demonstrate that the DAP-induced apoptosis of CIA-FLS occurred mainly via a caspase-dependent pathway, in particular the mitochondrial pathway, and that the Bax/Bcl-2 ratio was involved in this process. Thus, DAP may be a potential therapeutic agent for RA.

The potential target of double negative T cells in cancer immunotherapy.

e15180 Background: Double negative T cell (DNT) is defined by the expression of CD3 (or TCRαβ) with the absence of CD4, CD8 and iNKT cell markers. DNTs account for 1-3% of the total peripheral lymphocytes. Although healthy donor cells derived allogeneic DNTs were reported to have anti-tumor effects in several cancer models, the function of DNTs in tumor microenvironment is not well understood. In this study, we aim to characterize DNTs in cancer tissues and lymph nodes of colorectal cancer patients including their immunological functions. Methods: A total of 14 colorectal cancer tissues and regional lymph nodes were collected and examined for the proportion and the immunological functions of DNTs by FACS analysis. Results: DNTs, which were defined in this study as TCRαβ positive with CD4-, CD8- and CD56- T cells, were detected in colorectal cancer tissues. DNT cells account for more than 5% of TCRαβ-positive populations in 12 cases and out of which 7 cases were more than 20% observed in this study. In all of these 7 cases, DNT cells were also found to be higher in the tumor tissues than in non-metastatic lymph nodes. In addition to colorectal cancer, DNTs were found to be more than 5% of TCRαβ-positive populations in cancer tissues in 3 out of the 6 cases of ovarian and uterine cancer cases.To further evaluate the potential immunological functions of DNTs, expression of cell membrane protein markers were evaluated. CD3ε expression was down regulated in almost all of the DNTs. The CD107a expression of DNTs were higher than that of CD8+ T cells in tumor tissues. When co-culturing DNTs with tumor cells, CD107a expression of DNTs was significantly upregulated. Notably, the intracellular production of perforin and granzyme B were lower than CD8+ T cells. Fas-L expression were found to be higher on DNT cells compared to other T cell populations, suggesting DNT might possess cytotoxic activity in the tumor microenvironment. It is known that upregulation of Fas expression is caused by T cell activation and subsequently induces T cell apoptosis. The upregulation of Fas expression was also observed in DNT cells from non-metastatic lymph nodes. In contrast, Fas expression of DNT cells in tumor tissues was observed to be lower than CD4+ or CD8+ T cells, suggesting that DNT could escape from apoptosis. This downregulation of Fas expression was speculated to be one of the reasons for the accumulation of DNTs in tumor tissues. Conclusions: Although the function of DNTs was still not well elucidated, the accumulation of DNT cells in tumor microenvironment suggests DNTs could be a potential target for cancer immunotherapy.

Aptamer-T Cell Targeted Therapy for Tumor Treatment Using Sugar Metabolism and Click Chemistry.

The development of a tumor-targeted immunotherapy is highly required. The most advanced application is the use of CD19 chimeric antigen receptor (CAR)T (CAR-T) cells to B cell malignancies, but there are still side effects including potential carcinogenicity of lentiviral or retroviral insertion into the host cell genome. Here, we developed a nonviral aptamer-T cell targeted strategy for tumor therapy. Tumor cells surface-specific ssDNA aptamers were conjugated to CD3+T cells (aptamer-T cells) using N-azidomannosamine (ManNAz) sugar metabolic cell labeling and click chemistry. We found that the aptamer-T cells could specifically target and bind to tumor cells (such as SGC-7901 gastric cancer cell and CT26 colon carcinoma cell) in vitro and in mice after adoptively transfer in. Aptamer-T cells led to significant regression in tumor volume due to being enriched at tumor microenvironment and producing strong cytotoxicity activities of CD3+T cells with enhanced perforin, granzyme B, CD107a, CD69, and FasL expression. Moreover, aptamer-T displayed even stronger antitumor effects than an anti-PD1 immune-checkpoint monoclonal antibody (mAb) treatment in mice and combination with anti-PD1 yielded synergic antitumor effects. This study uncovers the strong potential of the adoptive nonviral aptamer-T cell strategy as a feasible and efficacious approach for tumor-targeted immunotherapy application.

Comparative gene expression analysis of Fas and related genes in preeclamptic and healthy women: A cross-sectional study.

BackgroundPreeclampsia is a hypertensive disorder of pregnancy affecting about 2-10% pregnancies worldwide. mRNA expression of tumor necrosis factor alpha (TNF-), Fas, and FasL have been reported to be altered in placental bed in preeclamptic pregnancies. We hypothesized that the expression of these genes is also altered in peripheral blood mononuclear cells (PBMCs) in preeclampsia.ObjectiveTo compare the expression of Fas receptor and related genes in PBMCs of preeclamptic and normotensive pregnant women.Materials and MethodsA cross-sectional comparative study comprising of 18 cases and 18 controls was designed. 5 ml of venous blood was drawn and collected considering aseptic measures. Buffy coat was separated by centrifugation and stored at -20°C. Favor Prep total RNA Isolation Kit (Favorgen, Taiwan) was used for RNA extraction. The mRNA expression of TNF-, Fas, and FasL was measured by real-time polymerase chain reaction in PBMCs in preeclamptic and normal pregnancies.ResultsA significant increase in mRNA expression of TNF-, Fas, and FasL (p 0.001) was observed in PBMCs of preeclamptic pregnancies compared to the control group (p 0.001). Moreover, a significant positive correlation was found between the TNF- mRNA expression and Fas and FasL (p 0.001).ConclusionThe results lead to the conclusion that mRNA expression of TNF-, Fas, and FasL in the maternal PBMCs is altered in preeclamptic pregnancies and might contribute to the pathogenesis of the disease.

Immunophenotypic and functional characterisation of Mesodermal Killer (MK) cells: a novel cell type and potential cellular therapy for cancer

Background & Aim We have identified a novel cell type, named mesodermal killer (MK) cells, derived from adult bone marrow under proprietary culture conditions. This study characterized the MK cells by immunophenotype, cytokine profile, immunomodulatory function and cytotoxicity. Methods, Results & Conclusion Methods: We investigated MK cells before and after activation with TNFα or IFNγ for immunophenotype using high throughput-FACS, for secretome and for inflammatory response in the murine air pouch assay. We evaluated MK cell cytotoxicity using the chromium release assay and immunomodulatory function by co-culture with natural killer (NK) cells in transwells. We used immunocytochemistry, flow cytometry and RT-PCR to detect expression of granzyme/perforin genes and proteins and investigated death pathways by siRNA silencing of FasL and TRAIL. Results MK cells displayed a novel immunophenotype (and different from BM-MSCs) including expression of CD16, CD96, CD112, CD137L, FasL, TRAIL and CD277 and some NK cell markers. In contrast to NK cells, MK cells lacked CD56 expression. TNFα and IFNγ activation increased FasL expression. MK cells secreted GROa, IL-12, IL-2Ra, IL-8, TRAIL and IL-6, and GROa and IL-8 secretion was increased by TNFα. MK cell cytotoxicity was observed against leukemia (K562), multiple myeloma (RPMI-8226) and breast cancer (MCF7) cell lines. MK cell conditioned media had no effect on MCF7 and antibody dependent cell mediated cytotoxicity (ADCC) experiments also proved negative. MK cells expressed granzymes and perforin and these were upregulated following exposure to RPMI-8226 (in contrast to BM-MSCs). Pre-treatment of MK cells with EGTA (a non-specific inhibitor of granule exocytosis) demonstrated >50% decrease in cytotoxicity against MCF7 suggesting a partial role of granule exocytosis in MK cell cytotoxicity. Inhibition of TRAIL expression by siRNA led to a significant reduction in MK cell cytotoxicity suggesting TRAIL may also be involved. In contrast to BM-MSCs, MK cells enhanced the cytotoxicity of primary NK cells against K562, RPMI-8226 and U226 (plasma cell myeloma) and IFNγ-activated MK cells enhanced migration of NK cells and monocytes into the murine air pouch suggesting a pro-inflammatory response. Conclusion MK cells exhibit cytotoxicity against a variety of cancer cell lines, are capable of priming NK cells in vitro and are pro-inflammatory in vivo. These results suggest the clinical translation of the MK cells is warranted and animal efficacy studies will begin shortly.

Inducing inflammatory response in RAW264.7 and NK-92 cells by an arabinogalactan isolated from Ferula gummosa via NF-κB and MAPK signaling pathways

The polysaccharide isolated from F. gummosa (FGP) was found homogenous with a weight average molecular weight (Mw) of 50.0 × 103 g/mol and radius of gyration (Rg) of 105.3 nm. The FGP was an arabinogalactan with a backbone formed of →6)-β-Galp-1→ residues having random branching points at C-3 extended with either β-Galp-(1→3)-β-Galp-(1→ or α-Araf-(1→ side chain residues. FGP exhibited proliferative effect on RAW264.7 cells and induced macrophages to exert proinflammatory response releasing NO and up-regulating the transcription of cytokines including TNF-α, IL-1β, IL-6 and IL-12. The FGP induced NK-92 cells to up-regulate the expressions of TNF-α, IFN-γ, granzyme-B, perforin, NKG2D and FasL. The presence of p-NF- κB, p-ERK, p-JNK and p-p38 in RAW264.7 and NK-92 cells indicated their activation through NF-κB and MAPKs signaling pathways. These findings suggested that polysaccharides from F. gummosa are potent in boosting immune system and thus may be considered for further studies of biomedical applications.

Characterization of the Impact of Daclizumab Beta on Circulating Natural Killer Cells by Mass Cytometry.

Daclizumab beta is a humanized monoclonal antibody that binds to CD25 and selectively inhibits high-affinity IL-2 receptor signaling. As a former treatment for relapsing forms of multiple sclerosis (RMS), daclizumab beta induces robust expansion of the CD56bright subpopulation of NK cells that is correlated with the drug’s therapeutic effects. As NK cells represent a heterogeneous population of lymphocytes with a range of phenotypes and functions, the goal of this study was to better understand how daclizumab beta altered the NK cell repertoire to provide further insight into the possible mechanism(s) of action in RMS. We used mass cytometry to evaluate expression patterns of NK cell markers and provide a comprehensive assessment of the NK cell repertoire in individuals with RMS treated with daclizumab beta or placebo over the course of 1 year. Treatment with daclizumab beta significantly altered the NK cell repertoire compared to placebo treatment. As previously reported, daclizumab beta significantly increased expression of CD56 on total NK cells. Within the CD56bright NK cells, treatment was associated with multiple phenotypic changes, including increased expression of NKG2A and NKp44, and diminished expression of CD244, CD57, and NKp46. These alterations occurred broadly across the CD56bright population, and were not associated with a specific subset of CD56bright NK cells. While the changes were less dramatic, CD56dim NK cells responded distinctly to daclizumab beta treatment, with higher expression of CD2 and NKG2A, and lower expression of FAS-L, HLA-DR, NTB-A, NKp30, and Perforin. Together, these data indicate that the expanded CD56bright NK cells share features of both immature and mature NK cells. These findings show that daclizumab beta treatment is associated with unique changes in NK cells that may enhance their ability to kill autoreactive T cells or to exert immunomodulatory functions.

Edaravone inhibits procaspase-3 denitrosylation and activation through FasL-Trx2 pathway in KA-induced seizure.

Previous studies have demonstrated that excessive free radicals play an essential role in the initiation and progression of epilepsy and that a novel exogenous free radical scavenger edaravone (Ed) exerts some neuroprotective effects on seizure-induced neuronal damage. The purpose of this study was to elucidate the possible molecular mechanisms of Ed associated with procaspase-3 denitrosylation and activation through the FasL-Trx2 pathway in seizures rats. In this study, we investigated the effects of Ed on the regulation of the combination of Fas ligand/Fas receptor and the major components of the death-inducing signaling complex (DISC) in the hippocampus of kainic acid (KA)-treated Sprague Dawley (SD) rats. Treatment with Ed can attenuate the increased expression of FasL induced by KA and prevent procaspase-3 denitrosylation and activation via suppression of the FasL-Trx2 signaling pathway, which alleviates the neuronal damage in seizures. These results provide experimental evidence that Ed functions by preventing the denitrosylation and activation of procaspase-3 and that Ed acts as a therapeutic option for epilepsy.

Differential Activation of Unconventional T Cells, Including iNKT Cells, in Alcohol-Related Liver Disease.

Liver is enriched in several innate-like unconventional T cells, but their role in alcohol-related liver disease (ALD) is not fully understood. Studies in several acute alcohol feeding models but not in chronic alcoholic steatohepatitis (ASH) model have shown that invariant natural killer T (iNKT) cells play a pathogenic role in ALD. Here, we investigated the activation of iNKT cells in an intragastric (iG) infusion model of chronic ASH as well as the frequency and cytokine phenotype of 3 different unconventional T cells: iNKT, mucosal-associated invariant T (MAIT), and CD8+ CD161hi Vα7.2- cells in peripheral blood of ALD patients. Hepatic iNKT cells were investigated using the iG model of chronic ASH that combines feeding of high-cholesterol/high-fat diet (HCFD) with intragastric feeding of ethanol diet (HCFD+iG Alc). Human iNKT, MAIT, and CD8+ CD161hi Vα7.2- cells were examined by flow cytometry in peripheral blood of patients with severe alcoholic hepatitis (SAH) and chronic alcoholics (ChA) and compared with healthy controls. In the iG model of chronic ASH, IFNγ+ iNKT cells accumulate in their livers compared with pair-fed control mice and activated hepatic iNKT cells show high expression of Fas and FasL. Notably, IFNγ+ iNKT cells are also significantly increased in peripheral blood of ChA patients compared with SAH patients. MAIT cells are significantly reduced in all ALD patients, but CD8+ CD161hi Vα7.2- cells are increased in SAH patients. Although MAIT and CD8+ CD161hi Vα7.2- cells displayed a similar cytokine production profile, the production of IFNγ and TNFα is significantly increased in SAH patients, while significant IL-17A production is found in ChA patients. We found that the 3 unconventional T cells are activated in ALD patients showing interesting differences in their frequency and cytokine production profile between SAH and ChA patients. In the iG murine model of chronic ASH, iNKT cells are also activated secreting proinflammatory cytokines suggesting their involvement in liver disease.

Evaluation of serum-free media formulations in feeder cell–stimulated expansion of natural killer cells

BackgroundOptimal expansion of therapeutic natural killer (NK) cell products has required media supplementation with human or fetal bovine serum, which raises safety and regulatory concerns for clinical manufacturing. Serum-free media (SFM) have been optimized for T-cell expansion, but few SFM systems have been developed for NK cells. Here, we compare six commercial clinical-grade SFM with our standard fetal bovine serum–containing medium for their ability to support NK cell expansion and function. MethodsHuman peripheral blood NK cells were expanded in selected media by recursive weekly stimulation with K562-based feeder cells expressing membrane-bound interleukin-21 and CD137L. Expansion was the primary readout, and the best-performing SFM was then compared with standard medium for cytotoxicity, phenotype, degranulation and cytokine secretion. Multiple lots were compared for consistency, and media was analyzed throughout for nutrient consumption and metabolic byproducts. ResultsTexMACS, OpTmizer, SCGM, ABS-001 and StemXVivo demonstrated equal or inferior NK cell expansion kinetics compared with standard medium, but expansion was markedly superior with AIM V + 5% Immune Cell Serum Replacement (ICSR; mean 5448 vs. 2621-fold expansion in 14 days). Surprisingly, NK cells expanded in AIM V + ICSR also showed increased cytotoxicity, tumor necrosis factor α secretion and DNAM-1, NKG2D, NKp30, FasL, granzyme B and perforin expression. Lot-to-lot variability was minimal. Glucose and glutamine consumption were inversely related to lactate and ammonia production. DiscussionThe AIM V + ICSR SFM system supports excellent ex vivo expansion of clinical-grade NK cells with the phenotype and function needed for adoptive immunotherapy.

Abstract A75: The role of eosinophils in the lung tumor microenvironment

Abstract Background: The use of immunotherapy to treat lung cancer is becoming increasingly common, highlighting the importance of the immune system in the lung tumor microenvironment. The lungs are host to a variety of immune cell subsets, including eosinophils (Eo), which are a population of innate immune cells that exert cytotoxic effector functions through the release of secretory granules and participate in tissue homeostasis and immunity. Despite the presence of Eo in solid tumors and their prevalence in the lung, the role of Eo in lung cancer is both controversial and largely unexplored. The Bennewith lab has previously found that mice with elevated lung Eo have decreased tumor growth in a model of breast cancer lung metastasis. We hypothesize that Eo play a protective role in lung cancer progression. Methods: In collaboration with Dr. Kelly McNagny (UBC), we used IL-5Tg transgenic mice that overexpress IL-5 and have a systemic expansion of Eo, ddGATA transgenic mice which are Eo-deficient, and ddGATA/IL-5Tg double-transgenic mice (excess IL-5 but no Eo) to study lung cancer. Lewis Lung carcinoma (LLC) cells were injected intravenously (IV) to seed the lungs. After three weeks, we harvested lungs and used flow cytometry to quantify immune cell subsets in the lungs. Additionally, we used clonogenic assays and histology to quantify lung tumor growth. Results: We confirmed that naive ddGATA and ddGATA/IL-5Tg mice have no lung Eo. In contrast, IL-5Tg mice have a 100-fold expansion of Eo in the lungs, and these Eo express higher levels of the Eo activation marker CD11b compared to wild-type (WT) mice. Naive IL-5Tg and ddGATA/IL-5Tg mice had an increased proportion of lung B-1 B cells, as well as an increase in the expression of the apoptosis-inducing cell surface molecule FasL. The absence of Eo in ddGATA mice did not impact lung colonization of LLC cells. Though there was a substantial expansion of Eo in the lungs of IL-5Tg mice compared to WT mice, there was no change in the number of lung-infiltrating Eo three weeks after IL-5Tg and WT mice were injected IV with LLC cells. IL-5Tg mice injected IV with LLC cells had an increase in the total number of lung-infiltrating Bconv and B-1 B cells compared to naive mice, whereas there was no change in B cell subsets between naive and LLC IV injected WT mice. Conclusions: Though Eo may play an antitumorigenic role in the presence of excess IL-5, the absence of Eo in ddGATA mice did not result in an increase in lung tumor burden. This suggests that Eo need to be activated and expanded to exert an antitumorigenic effect, or that the expansion of B cells in IL-5Tg mice is responsible for the decrease in lung tumor growth in IL-5Tg mice relative to WT mice. Illuminating the specific roles Eo and B cells play in lung cancer progression will allow us to better understand the interplay between host immune cells and malignant cells and could reveal new avenues of cancer immunotherapy development. Citation Format: Rachel A. Cederberg, Alvina So, Elizabeth Franks, Jenna Collier, Brennan J. Wadsworth, Michael R. Hughes, Kelly M. McNagny, Kevin L. Bennewith. The role of eosinophils in the lung tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A75.

LncRNA MAGI2-AS3 is down-regulated in intervertebral disc degeneration and participates in the regulation of FasL expression in nucleus pulposus cells

BackgroundIt is known that Fas ligand (FasL) is involved in the development of intervertebral disc degeneration (IDD). A recent study reported that lncRNA MAGI2-AS3 up-regulated the expression of FasL to promote breast cancer. Therefore, we investigated the roles that lncRNA MAGI2-AS3 might play in IDD.MethodsA total of 66 IDD patients (IDD group) and 58 healthy volunteers (Control group) were recruited in this study. Quantitative real-time PCR (qRT-PCR) and western blot were used to investigate gene expression levels. Cell transfections were carried out to analyze gene interactions. The diagnostic value of lncRNA MAGI2-AS3 for IDD was assessed by ROC curve analysis.ResultsThe expression levels of plasma lncRNA MAGI2-AS3 were lower in IDD patients compared to that in the control group. Down-regulation of lncRNA MAGI2-AS3 effectively distinguished IDD patients from the control group. The expression levels of plasma lncRNA MAGI2-AS3 were significantly increased after the treatments. Over-expression of lncRNA MAGI2-AS3 inhibited the expression of FasL, while the silencing of lncRNA MAGI2-AS3 promoted the expression of FasL in nucleus pulposus (NP) cells.ConclusionsTherefore, lncRNA MAGI2-AS3 is down-regulated in IDD and participates in the regulation of FasL expression in nucleus pulposus (NP) cells.

Long noncoding RNA LINC00324 exerts protumorigenic effects on liver cancer stem cells by upregulating fas ligand via PU box binding protein.

Hepatocellular carcinoma (HCC) represents a major cause of cancer death, but the molecular mechanism for its development has not yet been well characterized. Long noncoding RNAs (lncRNAs) are involved in a wide range of biological processes via their roles as oncogenes or tumor suppressor genes. The present study aimed to elucidate the role of LINC00324 in HCC through its interaction with Fas ligand (FasL). Initially, microarray-based gene expression profiling of HCC was employed to identify differentially expressed genes. Next, the expression of LINC00324 in HCC tissues and liver cancer stem cell (LCSC) lines was examined using RT-qPCR. Then, the interaction among LINC00324, PU box binding protein (PU.1) and FasL was identified with RIP, ChIP and dual-luciferase reporter gene assays. The effect of LINC00324 on viability, proliferation, migration, invasion, and apoptosis as well as the tumorigenesis of transfected cells was examined with gain- and loss-of-function experiments. LINC00324 and FasL were highly expressed in HCC. LINC00324 regulated FasL expression via interaction with PU.1. Silencing of LINC00324 or FasL suppressed expression of stemness-related genes, cell viability, proliferation, migration, invasion, self-renewal, and tumorigenesis, but enhanced cell apoptosis. Taken together, LINC00324 promotes the expression of FasL through the recruitment of PU.1, which ultimately maintains the biological properties of LCSCs, thus, highlighting LINC00324 as a promising therapeutic candidate for HCC.

Fas Ligand (FasL) in Association with Tumor-Infiltrating Lymphocytes (TILs) in Early Stage Cervical Cancer.

Objective:To date, little is known about the roles of FasL and TILs in cervical cancer. This study aims to determine the correlation between FasL expression and TILs presence in cervical cancer. Methods:In this study, we analysed the FasL and TIL presence in 32 squamous cell carcinoma or adenocarcinoma that were obtained from early stage (≤ IIA2) cervical cancer patients using immunohistochemistry. The level of FasL and TIL was assessed qualitatively, and then quantified with the H-Score system. Results:Most of the patients were between 30 to 50 years old (59,4%), and had never taken pap smear examination before (96,9%). Based on the Pearson analysis of FasL and TIL presence, we found that FasL was inversely correlated with CD45 or TIL number when the level of FasL is above 140 and the CD45 is below 160. Based on Chi-Square test of FasL and TIL classification, there was a nine-fold odds ratio (OR) of lower TILs classification in high expression of FasL classification (OR 9, p=0.01). Conclusion:An inverse correlation between FasL expression and TILs level, that might indicate FasL-induced TILs apoptosis in tumor tissue, was observed. The strong inverse correlation between FasL and TILs presence showed some insight about the interactions between cancer cells and its surroundings inside of the cervical cancer tissue. This might also be further developed to tailor a prognostic marker that can predict the outcome of therapy in patients, not only in cervical cancer, but generally in all cancer.