TRAIL-dependent Manner Research Articles

HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

IFN-β activates cytotoxic function of human natural killer cells toward IL-27 and poly(I:C) stimulated PC3 and DU145 cells

Natural killer (NK) cell phenotype and function are altered in patients with prostate cancer, and increased NK cell activity is associated with a better prognosis in patients with disease. For patients with advanced stage prostate cancer, immunotherapies are a promising approach when standard treatment options have been exhausted. With the rapid emergence of NK cell-based therapies, it is important to understand the mechanisms by which NK cells can be triggered to kill cancer cells that have developed immune-evasive strategies. Altering the cytokine profiles of advanced prostate cancer cells may be an area to explore when considering ways in which NK cell activation can be modulated. We have previously demonstrated that combining the cytokine, IL-27, with TLR3 agonist, poly(I:C), changes cytokine secretion in the advanced prostate cancer models, PC3 and DU145 cells. Herein, we extend our previous work to study the effect of primary human NK cells on prostate cancer cell death in an in vitro co-culture model. Stimulating PC3 and DU145 cells with IL-27 and poly(I:C) induced IFN-β secretion, which was required for activation of primary human NK cells to kill these stimulated prostate cancer cells. PC3 cells were more sensitized to NK cell-mediated killing when compared to DU145 cells, which was attributed to differential levels of IFN-β produced in response to stimulation with IL-27 and poly(I:C). IFN-β increased granzyme B secretion and membrane-bound TRAIL expression by co-cultured NK cells. We further demonstrated that these NK cells killed PC3 cells in a partially TRAIL-dependent manner. This work provides mechanistic insight into how the cytotoxic function of NK cells can be improved to target cancer cells.

Transferrin-Bound Doxorubicin Enhances Apoptosis and DNA Damage through the Generation of Pro-Inflammatory Responses in Human Leukemia Cells.

Doxorubicin (DOX) is an effective antineoplastic drug against many solid tumors and hematological malignancies. However, the clinical use of DOX is limited, because of its unspecific mode of action. Since leukemia cells overexpress transferrin (Tf) receptors on their surface, we proposed doxorubicin–transferrin (DOX–Tf) conjugate as a new vehicle to increase drug concentration directly in cancer cells. The data obtained after experiments performed on K562 and CCRF-CEM human leukemia cell lines clearly indicate severe cytotoxic and genotoxic properties of the conjugate drug. On the other hand, normal peripheral blood mononuclear cells (PBMCs) were more resistant to DOX–Tf than to DOX. In comparison to free drug, we observed that Tf-bound DOX induced apoptosis in a TRAIL-dependent manner and caused DNA damage typical of programmed cell death. These fatal hallmarks of cell death were confirmed upon morphological observation of cells incubated with DOX or DOX–Tf. Studies of expression of TNF-α, IL-4, and IL-6 at the mRNA and protein levels revealed that the pro-inflammatory response plays an important role in the toxicity of the conjugate. Altogether, the results demonstrated here describe a mechanism of the antitumor activity of the DOX–Tf conjugate.

M1 Macrophages Promote TRAIL Expression in Adipose Tissue-Derived Stem Cells, Which Suppresses Colitis-Associated Colon Cancer by Increasing Apoptosis of CD133+ Cancer Stem Cells and Decreasing M2 Macrophage Population.

We have previously reported that adipose tissue-derived stem cells (ASCs) cultured at high cell density can induce cancer cell death through the expression of type I interferons and tumor necrosis factor (TNF)-related apoptosis-inducing ligands (TRAIL). Here, we investigated whether TRAIL-expressing ASCs induced by M1 macrophages can alleviate colitis-associated cancer in an azoxymethane (AOM)/dextran sodium sulfate (DSS) animal model. M1 macrophages significantly increased the TRAIL expression in ASCs, which induced the apoptosis of LoVo cells in a TRAIL-dependent manner. However, CD133knockout LoVo cells, generated using the CRISPR-Cas9 gene-editing system, were resistant to TRAIL. In the AOM/DSS-induced colitis-associated cancer model, the intraperitoneal transplantation of TRAIL-expressing ASCs significantly suppressed colon cancer development. Moreover, immunohistochemical staining revealed a low CD133 expression in tumors from the AOM/DSS + ASCs group when compared with tumors from the untreated group. Additionally, the ASC treatment selectively reduced the number of M2 macrophages in tumoral (45.7 ± 4.2) and non-tumoral mucosa (30.3 ± 1.5) in AOM/DSS + ASCs-treated animals relative to those in the untreated group (tumor 71.7 ± 11.2, non-tumor 94.3 ± 12.5; p < 0.001). Thus, TRAIL-expressing ASCs are promising agents for anti-tumor therapy, particularly to alleviate colon cancer by inducing the apoptosis of CD133+ cancer stem cells and decreasing the M2 macrophage population.

Abstract 258: Recombinant human TRAIL or a DR5 agonistic antibody convert the response of non-triple negative breast cancer cells to ONC201 from anti-proliferative to apoptotic

Abstract ONC201 was initially identified as an inducer of cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. The compound is being tested in patients with a variety of tumor types, including those of the breast. Both triple negative breast cancer (TNBC) cells and non-TNBC cells are sensitive to ONC201. In a subset of TRAIL-sensitive TNBC cells, ONC201 treatment leads to the induction of apoptosis in a TRAIL-dependent manner. In the majority of TRAIL-resistant non-TNBC cells, ONC201’s effects are anti-proliferative rather than apoptotic. In vivo, the apoptotic response to ONC201 leads to efficacy of the compound while the anti-proliferative response does not (Ralff et al., Mol Cancer Ther., 2017). Here we worked to identify strategies to convert the response of cancer cells to ONC201 from anti-proliferative to apoptotic. ONC201 treatment primed TRAIL-resistant non-TNBC cells to undergo TRAIL-dependent cell death. Increases in the mRNA and surface protein levels of death receptor 5 (DR5) as well as decreases in the expression of multiple anti-apoptotic proteins occured in ONC201-treated cells. We also observed small but significant increases in TRAIL mRNA and surface protein in non-TNBC cells following treatment with ONC201. Despite apoptotic priming and the induction of TRAIL, the effect of the compound in the non-TNBC cells remains anti-proliferative but not apoptotic. We hypothesized that the level of TRAIL induced by ONC201 in these cells, known to be TRAIL-resistant, was insufficient. Remarkably, the addition of exogenous recombinant human TRAIL (rhTRAIL) converted the response of TRAIL-resistant non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Cleaved PARP, caspase-3, caspase-9, and caspase-8 were observed in cells treated with ONC201 followed by rhTRAIL, but not in cells treated with ONC201 or rhTRAIL alone. Propidium iodide staining and quantification of the subG1 population via flow cytometry further confirmed these findings. Similar observations about the ability of rhTRAIL to convert the cellular response to ONC201 from anti-proliferative to apoptotic were made in cancer cells from other tumor types. Importantly, rhTRAIL did not convert the response of normal fibroblasts to ONC201 from anti-proliferative to apoptotic. Addition of a DR5 agonistic antibody to ONC201 treated non-TNBC cells also converted the response of the non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Ongoing experiments are currently testing the combination of ONC201 and rhTRAIL in vivo. These findings describe a strategy for potently converting the response of a cancer cell to ONC201 from anti-proliferative to apoptotic. Clinical application of this combination therapy may result in tumor regressions in patients with decreased sensitivity to ONC201 as a single agent. Citation Format: Marie D. Ralff, Jocelyn E. Ray, Avital Lev, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Recombinant human TRAIL or a DR5 agonistic antibody convert the response of non-triple negative breast cancer cells to ONC201 from anti-proliferative to apoptotic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 258.

KLRG1+ natural killer cells exert a novel antifibrotic function in chronic hepatitis B

Natural killer (NK) cells are known to exert strong antiviral activity. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is expressed by terminally differentiated NK cells and KLRG1-expressing lymphocytes are known to expand following chronic viral infections. We aimed to elucidate the previously unknown role of KLRG1 in the pathogenesis of chronic hepatitis B (CHB). KLRG1+ NK cells were taken from the blood and liver of healthy individuals and patients with CHB. The phenotype and function of these cells was assessed using flow cytometry and in vitro stimulation. Patients with CHB had a higher frequency of KLRG1+ NK cells compared to healthy controls (blood 13.4 vs. 2.3%, p <0.0001 and liver 23.4 vs. 2.6%, p <0.01). KLRG1+ NK cells were less responsive to K562 and cytokine stimulation, but demonstrated enhanced cytotoxicity (9.0 vs. 4.8%, p <0.05) and IFN-γ release (8.0 vs. 1.5%, p <0.05) via antibody dependent cellular cytotoxicity compared to their KLRG1- counterparts. KLRG1+ NK cells possessed a mature phenotype, demonstrating stronger cytolytic activity and IFN-γ secretion against hepatic stellate cells (HSCs) than KLRG1- NK cells. Moreover, KLRG1+ NK cells more effectively induced primary HSC apoptosis in a TRAIL-dependent manner. Increased KLRG1+ NK cell frequency in the liver and blood was associated with lower fibrosis stage (F0/F1) in patients with CHB. Finally, the expression of CD44, degranulation and IFN-γ production were all increased in KLRG1+ NK cells following stimulation with osteopontin, the CD44 ligand, suggesting that HSC-derived osteopontin may cause KLRG1+ NK cell activation. KLRG1+ NK cells likely play an antifibrotic role during the natural course of CHB infection. Harnessing this antifibrotic function may provide a novel therapeutic approach to treat liver fibrosis in patients with CHB. Individuals that are chronically infected with hepatitis B virus (HBV) possess an increased number of immune cells, called natural killer (NK) cells expressing the surface marker KLRG1 in the blood and liver. Here, we demonstrate that these specific NK cells are able to kill activated stellate cells in the liver. Because activated stellate cells contribute to liver scarring, i.e. fibrosis, and subsequent liver dysfunction in individuals with chronic HBV infection, KLRG1+ NK cells are a novel immune cell type that can limit liver scarring.

Low-Dose Radiation Conditioning Enables CAR T Cells to Mitigate Antigen Escape.

CD19 chimeric antigen receptors (CARs) have demonstrated great efficacy against a range of B cell malignancies. However, antigen escape and, more generally, heterogeneous antigen expression pose a challenge to applying CAR therapy to a wide range of cancers. We find that low-dose radiation sensitizes tumor cells to immune rejection by locally activated CAR Tcells. In a model of pancreatic adenocarcinoma heterogeneously expressing sialyl Lewis-A (sLeA), we show that not only sLeA+ but also sLeA- tumor cells exposed to low-dose radiation become susceptible to CAR therapy, reducing antigen-negative tumor relapse. RNA sequencing analysis of low-dose radiation-exposed tumors reveals the transcriptional signature of cells highly sensitive to TRAIL-mediated death. We find that sLeA-targeted CAR Tcells produce TRAIL upon engaging sLeA+ tumor cells, and eliminate sLeA- tumor cells previously exposed to systemic or local low-dose radiation in a TRAIL-dependent manner. These findings enhance the prospects for successfully applying CAR therapy to heterogeneous solid tumors. Local radiation is integral to many tumors' standard of care and can be easily implemented as a CAR conditioning regimen.

Abstract 3786: ILC-k: Human innate lymphoid cells displaying unique metabolic features and KIR-independent cytotoxicity, impaired in acute myeloid leukemia

Abstract Innate Lymphoid Cells (ILCs) are a recently identified family of lymphocytes, divided into 3 different groups that mirror the functional specialization of helper CD4 T cells. However, it is now clear that the spectrum of ILC diversity is much broader than originally appreciated. ILCs have been shown to play important roles in inflammatory and autoimmune diseases, and their involvement in anti-tumor responses has also recently been evidenced by us and others. Here, we report on the identification of a previously undescribed human ILC subset, named hereafter “ILC-killer” (ILC-k), characterized by a Lineage negative, CD127+ cKIT- CRTh2- CD56+ phenotype, thus being distinct from conventional helper ILCs, e.g ILC1 (CD56-), ILC2 (CRTh2) and ILC3 (cKIT+). Transcriptomic profiling of ex-vivo highly pure ILC-k, conventional ILC subsets and conventional Natural Killer (NK) cell subsets showed closeness between ILC-k, ILC3 and CD56bright NK cells. However, ILC-k display a specific metabolism (low glucose uptake, high mitochondrial activity) and cytotoxic gene signature, also confirmed at protein level. By their functional evaluation, ILC-k showed specific ability to kill tumor cell lines in a KIR-independent, but NKp30- and Trail-dependent manner. Their relative frequency is decreased and both their metabolic fitness and cytotoxic potential are impaired in Acute Myeloid Leukemia (AML) patients at diagnosis (N=51). Upon remission, ILC frequencies and functions are restored to normal levels. Overall, we identified cytotoxic ILCs, distinct from conventional NK cells, that are functionally impaired in AML. Given their KIR-independence, ILC-k might represent promising therapeutic candidates to improve NK-cell based immunotherapy, that currently necessitates KIR-HLA mismatch between donor and recipient for full efficiency. Citation Format: Bérengère Salomé, Alejandra Gomez-Cadena, Romain Loyon-Bonato, Madeleine Suffiotti, Valentina Salvestrini, Antonio Curti, Paolo Tentorio, Domenico Mavilio, Carsten Riether, Adrian Ochsenbein, Emanuela Marcenaro, David Gfeller, Pedro Romero, Sara Trabanelli, Camilla Jandus. ILC-k: Human innate lymphoid cells displaying unique metabolic features and KIR-independent cytotoxicity, impaired in acute myeloid leukemia [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 3786.

Abstract LB-082: ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation

Abstract ONC201 is a potent inducer of cancer cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. ONC201 and its structural analogs comprise a novel class known as the imipridones. The compound is being tested clinically against a range of solid tumors and hematological malignancies. We found that both triple negative and non-triple negative breast cancer cells are sensitive to ONC201. This was initially surprising, as the majority of non-triple negative breast cancer cells are resistant to TRAIL. In TRAIL-sensitive cells, ONC201 induced cell death in a TRAIL-dependent manner. In TRAIL-resistant cells, the effects of ONC201 are not pro-apoptotic but are rather anti-proliferative. Here we further investigate the effects of ONC201 in TRAIL-resistant breast cancers from multiple molecular subtypes. ONC201 has been previously shown to induce transcriptional upregulation of TRAIL receptor death receptor 5 (DR5). A known mechanism of TRAIL resistance in breast cancer is low cell surface DR5 expression. We hypothesized that pre-treatment with ONC201 would upregulate DR5 and lead to TRAIL sensitization in resistant cells. Our results confirm our hypothesis and show that treatment with ONC201 transcriptionally upregulates DR5 and increases its expression at the cell surface. In addition, pre-treatment with ONC201 sensitizes TRAIL-resistant breast cancer cells to the pro-apoptotic effects of TRAIL. Caspase-8 and PARP are cleaved when cells are treated with the combination of ONC201 and TRAIL, but not when treated with either compound alone. Annexin-V PI staining and quantitation of subG1 DNA content via flow cytometry further confirmed these results. Knockdown of DR5 abrogated TRAIL sensitization by ONC201. Natural killer (NK) cells are known to use TRAIL to kill targets like tumor cells. We are exploring the hypothesis that pre-treatment of tumor cells with ONC201 would increase their killing by NK cells. We are currently investigating the importance of the TRAIL pathway and DR5 upregulation by imipridone compounds in this sensitization to NK cell induced apoptosis. In summary, these results describe a novel mechanism by which ONC201 sensitizes breast cancer cells to the effects of apoptosis inducing ligand TRAIL. They also provide preclinical rationale for the testing of the compound against breast cancers in the clinic. Citation Format: Marie D. Ralff, Jessica Wagner, Wafik S. El-Deiry. ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation [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 LB-082.

STAT5 inhibition induces TRAIL/DR4 dependent apoptosis in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a rare, aggressive, heterogeneous, Non-Hodgkin's lymphoma with poor prognosis and inadequate response to current therapies. Recent sequencing studies indicate a prevalence of activating mutations in the JAK/STAT signaling pathway. Oncogenic mutations in STAT5B, observed in approximately one third of cases of multiple different PTCL subtypes, correlate with inferior patient outcomes. Therefore, interest in the development of therapeutic strategies for targeting STAT5 in PTCL is warranted. In this study, we show that the drug pimozide inhibits STAT5 in PTCL, leading to apoptotic cell death by means of the TRAIL/DR4 dependent extrinsic apoptotic pathway. Pimozide induced PTCL cell death is caspase 8 dependent, increases the expression of the TRAIL receptor, DR4, on the surface of pre-apoptotic PTCL cells, and enhances TRAIL induced apoptosis in a TRAIL dependent manner. In parallel, we show that mRNA and protein levels of intrinsic pathway BCL-2 family members and mitochondrial membrane potential remain unaffected by STAT5 knockdown and/or inhibition. In primary PTCL patient samples, pimozide inhibits STAT5 activation and induces apoptosis. Our data support a role for STAT5 inhibition in PTCL and implicate potential utility for inhibition of STAT5 and activation of the extrinsic apoptotic pathway as combination therapy in PTCL.

Abstract 3000: Intra-tumoral accumulation of NK1.1/CD3+ cells and anti-metastasis effects of dose-intensified ONC201 in tumor-bearing mice

Abstract ONC201, a novel first-in-class, orally active anti-tumor agent that upregulates the cytotoxic ligand TRAIL (Allen et al., Sci. Trans. Med., 2013; Wagner et al., Oncotarget, 2014), activates the integrated stress response leading to tumor cell upregulation of TRAIL death receptor 5 (Kline et al., Sci. Sig., in press). ONC201 is active against bulk tumor and cancer stem cells (Prabhu et al., Can. Res., 2015). ONC201 is under clinical development by Oncoceutics, and is being evaluated in multiple phase I/II clinical trials. Results of the first-in-human ONC201 study presented at the 2015 AACR-NCI-EORTC meeting (Stein et al., Abstract C138) demonstrated ONC201 to be safe in humans, to exhibit predicted PK and sustained PD characteristics, and revealed a preliminary efficacy signal. As patients were dosed on an every 3-week schedule, based on supportive preclinical data, we investigated dose-intensification of ONC201 to determine whether a higher dose/frequency schedule might impact efficacy with limited toxicity. We hypothesized that ONC201 may be effective in dose-intensified schedule and may inhibit metastases. We tested a range of ONC201 doses including 25, 50, and 100 mg/kg and frequencies including every 4, 3, 2, 1 week as well as twice a week dosing. In colon and triple-negative breast cancer we observed that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo. Frequency effects were more pronounced at lower doses and dose-dependency was more impactful with less frequent schedules. We noted a potent anti-metastasis effect of ONC201 in vivo, not previously reported, as a function of both increased ONC201 dose and frequency of administration. ONC201 inhibits cancer cell migration and invasion in vitro in a TRAIL-dependent manner. We found ONC201 more potently suppresses Akt and ERK in tumors in vivo in a dose- and frequency-dependent manner, whereas its effect on TRAIL serum levels appeared to be impacted by frequency. We observed accumulation of CD3+/NK1.1+ cells within ONC201-treated tumors in athymic nude mice that lack T-cells. Accumulation of CD3+/NK1.1 cells within ONC201-treated tumors was more pronounced with dose intensification that correlated with superior efficacy. In summary, we have uncovered a potent anti-metastasis effect of ONC201 coupled with the appearance of CD3+/NK1.1+ cells within ONC201-treated tumors. We are further evaluating the biomarker characteristics and immune function of the CD3+/NK1.1+ cells and the relationship of their intra-tumoral accumulation to observed anti-tumor effects of ONC201, including in fully immunocompetent mice. Our results suggest that clinical investigation of both dose and frequency of ONC201 administration is warranted and is being evaluated in an ongoing clinical trial (NCT02609230). Citation Format: Jessica Wagner, Christina L.B. Kline, Marie Baumeister, Wafik S. El-Deiry. Intra-tumoral accumulation of NK1.1/CD3+ cells and anti-metastasis effects of dose-intensified ONC201 in tumor-bearing mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3000.

5-Fluorouracil-induced RNA stress engages a TRAIL-DISC-dependent apoptosis axis facilitated by p53.

Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-treated p53-/- cells is demonstrated to be caspase-dependent, but due to a slow pace, accumulation of mitochondrial reactive oxygen species contributes to necrotic characteristics. The oligomerization status of the p53 target gene DR5 is determined as a significant limiting factor for the initiation of caspase activity in an intracellular TRAIL-dependent manner. Using several experimental approaches, we further conclude that RNA-rather than DNA-related stress follows by caspase activation irrespectively of p53 status. A distinct 5-FU-induced stress mechanism is thereby functionally connected to a successive and discrete cell death signaling pathway. Finally, we provide evidence that silencing of PARP-1 function may be an approach to specifically target p53-deficient cells in 5-FU combinatorial treatment strategies. Together, our results disclose details of impaired cell death signaling engaged as a consequence of 5-FU chemotherapy. Obtained data will contribute to the comprehension of factors restraining 5-FU efficiency, and by excluding DNA as the main stress target in some cell types they propose alternatives to currently used and suggested synergistic treatment regimens.

845 Glycosylation of MUC1 expressing on prostate cancer cell has crucial impact on susceptibility to natural killer cell immunity in TRAIL dependent manner

845 Glycosylation of MUC1 expressing on prostate cancer cell has crucial impact on susceptibility to natural killer cell immunity in TRAIL dependent manner

IL-27 Enhances the Expression of TRAIL and TLR3 in Human Melanomas and Inhibits Their Tumor Growth in Cooperation with a TLR3 Agonist Poly(I:C) Partly in a TRAIL-Dependent Manner

Interleukin (IL)-27 is a member of the IL-6/IL-12 cytokine family and possesses potent antitumor activity, which is mediated by multiple mechanisms. Toll-like receptor (TLR)3 is the critical sensor of the innate immune system that serves to identify viral double-stranded RNA. TLR3 is frequently expressed by various types of malignant cells, and recent studies reported that a synthetic TLR3 agonist, polyinosinic-polycytidylic acid [poly(I:C)], induces antitumor effects on malignant cells. In the present study, we have explored the effect of IL-27 on human melanomas and uncovered a previously unknown mechanism. We found that IL-27 inhibits in vitro tumor growth of human melanomas and greatly enhances the expression of TNF-related apoptosis inducing ligand (TRAIL) in a dose-dependent manner. Neutralizing antibody against TRAIL partly but significantly blocked the IL-27–mediated inhibition of tumor growth. In addition, IL-27 and poly(I:C) cooperatively augmented TRAIL expression and inhibited tumor growth. The cooperative effect could be ascribed to the augmented expression of TLR3, but not retinoic acid-inducible gene-I or anti-melanoma differentiation-associated gene 5, by IL-27. The inhibition of tumor growth by the combination was also significantly abrogated by anti-TRAIL neutralizing antibody. Moreover, IL-27 and poly(I:C) cooperatively suppressed in vivo tumor growth of human melanoma in immunodeficient mice. Taken together, these results suggest that IL-27 enhances the expression of TRAIL and TLR3 in human melanomas and inhibits their tumor growth in cooperation with poly(I:C), partly in a TRAIL-dependent manner. Thus, IL-27 and the combination of IL-27 and poly(I:C) may be attractive candidates for cancer immunotherapy.

Functional plasticity of regulatory T cells (168.7)

Abstract Regulatory T cells (Tregs) play a pivotal role in maintaining immune homeostasis, preventing autoimmunity, controlling exaggerated immune responses against infections and suppressing anti-tumor immunity. Tregs utilize multiple mechanisms to mediate their function. However, the number of mechanisms critical to exert maximal suppressive activity is unclear. It is conceivable that suppressive mechanisms could be tightly regulated by cross-regulatory networks. It has been shown that interleukin 10 (IL10) and IL35 have critical role in Treg function. Therefore, we assessed the functional capacity of Tregs lacking the ability to secrete IL10 and IL35. Surprisingly, our study revealed that Tregs lacking both IL10 and IL35 (Ebi3-/-Il10-/- or Il12a-/-Il10-/-) were fully functional and were able to suppress Tconv cells both in vitro and in vivo . Absence of IL10 and IL35 was compensated by an increase in cathepsin E (CTSE), influencing the release of soluble TRAIL and thereby resulting in suppression of target cells in a TRAIL-dependent manner. Inhibiting TRAIL activity disrupted the function of Ebi3-/-Il10-/- Tregs. The loss of suppressive function was maximal in Tregs lacking IL10, IL35 and TRAIL (Ebi3-/-Il10-/-Tnfsf10-/-Tregs). These data suggests the existence of cross-regulatory networks which controls the utilization of suppressive factors, thereby maintaining Treg functional plasticity.

789 PRECLINICAL CHARACTERIZATION OF NOVEL CYCLOPHILIN INHIBITORS BASED ON THE POLYKETIDE, SANGLIFEHRIN

Introduction: Progressive hepatic fibrosis is a common feature of chronic hepatitis C. Natural Killer (NK) cells represent a major component of intra-hepatic lymphocytes and have been shown to be important for the early control and natural course of HCV infection. Moreover, in mouse models it has been shown that natural killer cells can attenuate liver fibrosis via killing of activated hepatic stellate cells (HSC) in a NKG2D and TRAIL dependent manner. Here, we analyzed the interactions of human NK cells from HCVpositive patients with activated primary HSC and the potential effects of interferon-a (IFN-a) treatment. Material and methods: NK cells from untreated HCV-RNA(+) patients (n = 11), interferon-a (IFN-a) treated patients (n = 10) and healthy controls (n = 12) were co-incubated with activated primary HSC (ScienCell). NK cells from healthy persons were incubated in the presence or absence of IFN-a (25 IU/ml). Cytotoxic activity of NK cells was studied using the CD107a assay. In addition, INF-g and TNFa production of NK cells was measured by FACS analysis. Induction of HSC apoptosis (active Caspase-3) was analyzed flowcytometrically. Results: Analyzing cytolytic activity of NK cells following coincubation with HSC only discrete CD107a expression could be observed on both NK cells from HCV(+) patients and healthy controls ([mean±SEM: 5.7±0.7% vs. 4.7±0.8%; p =n.s.). Furthermore, only negligible secretion of IFN-g and TNF-a could be observed. However, NK cells from untreated HCV-infected patients were significantly more effective in induction of HSC apoptosis (20.8±2.1%) than NK cells from healthy controls (6.2±0.6%). Of note, NK cells from IFN-a/RBV treated HCV(+) patients displayed an even stronger capability to kill HSC (26.9±4.3%). In vitro stimulation of NK cells with IFN-a resulted in a significant up-regulation of TRAIL and was associated with increased killing of HSC as compared to un-stimulated NK cells. This effect could be blocked with both NKG2Dand TRAIL-specific antibodies. Conclusion: NK cells from HCV-infected patients are highly efficient in inducing apoptosis of activated hepatic stellate cells. This function of NK cell is increased following IFN-a treatment. Thus, NK cells may play an important anti-fibrotic role in chronic hepatitis C.

Systemic Immunological Tolerance to Ocular Antigens Is Mediated by TRAIL-Expressing CD8+ T Cells

Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required Fas ligand-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8(+) regulatory T cells. This study examined the mechanism by which these CD8(+) regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4(+) T cells and led to increased TRAIL expression by splenic CD8(+) T cells. Unlike wild-type mice, Trail(-/-) or Dr5(-/-) mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8(+) T cells from Trail(-/-) mice that were first injected via the AC with Ag were unable to transfer tolerance to naive recipient wild-type mice, but CD8(+) T cells from AC-injected wild-type or Dr5(-/-) mice could transfer tolerance. Importantly, the transferred wild-type (Trail(+/+)) CD8(+) T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail(-/-) CD8(+) T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that "helpless" CD8(+) regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.

The role of c-FLIP L in ovarian cancer: Chaperoning tumor cells from immunosurveillance and increasing their invasive potential

Objective In the current study, we aimed to investigate the role of the long isoform of cellular Fas-associated death domain-like interleukin-1β-converting enzyme (FLICE)-like inhibitory protein (c-FLIP L) in ovarian cancer (OC) development by using RNA interference (RNAi) in vitro and in vivo. Methods TRAIL-resistant human OC cell lines were genetically manipulated by RNAi-mediated suppression of c-FLIP L. Subsequently, the genetic alteration that was introduced into the various OC cell lines was characterized in vitro and in vivo. Results We previously showed that about 40% of OC patients express high levels of c-FLIP L, and that natural killer (NK) cells mediated immunosurveillance in OC. In the present study, we observed that the knockdown of c-FLIP L in human OC cell lines not only enhanced their sensitivity to TRAIL-mediated apoptosis, but also inhibited their migratory phenotype in a TRAIL-dependent manner in vitro. Shutdown of c-FLIP L in OC cells significantly decreased tumor development by induction of apoptosis and reduction of proliferation in vivo. Importantly, the knockdown of c-FLIP L particularly inhibited the invasion of OC cells into the peritoneal cavity, which might be due to high expression of TRAIL by NK cells and NK-cell mediated immunosurveillance. Conclusion These data demonstrate that c-FLIP L exhibits multiple functions in OC cells: first by concomitantly evading the natural immunity mediated by TRAIL-induced cell death, and second by augmenting cell motility and invasion in vivo. Our findings indicate that c-FLIP L regulates sensitivity of OC to TRAIL-mediated apoptosis and offers possible therapeutical implications for OC in the future.

TRAIL-expressing CD8+ regulatory T cells participate in the maintenance of ocular immune privilege (39.40)

Abstract Immunological tolerance to antigens encountered in the eye, the prototypic immune privileged site, inhibits potentially damaging immune responses that would otherwise be initiated to these antigens at other anatomical locations. The induction of tolerance depends on the FasL-mediated apoptotic death of inflammatory cells that enter the eye in response to antigenic challenge. Moreover, the tolerance is not due to the failure to prime, but is mediated by the generation of CD8+ regulatory T cells (Treg) that can transfer tolerance to a naïve recipient. The present study investigated the mechanism of action of the induced CD8+ Treg. Unlike wildtype (WT) mice, TRAIL-/- or DR5-/- mice did not develop tolerance to antigen injected into the eye, even though the responding lymphocytes underwent apoptosis in the eyes of all mice. We also found that splenocytes from tolerant WT mice produced significantly less IFN, compared to TRAIL-/- mice, after restimulation. Interestingly, CD8+ T cells from TRAIL-/- mice were unable to transfer tolerance to naïve recipient WT mice, but CD8+ T cells from WT or DR5-/- mice could transfer tolerance. Subsequent investigation found that tolerant animals contained CD8+ Treg cells capable of killing antigen-pulsed splenocytes via TRAIL. Together, our data indicate that CD8+ Treg enforce tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.

In Chronic Pancreatitis, Widespread Emergence of TRAIL Receptors in Epithelia Coincides with Neoexpression of TRAIL by Pancreatic Stellate Cells of Early Fibrotic Areas

TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis by cross-linking of the two TRAIL receptors that contain a death domain, TRAIL-R1 and TRAIL-R2. TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. Our aim was to determine the expression of TRAIL and its receptors in normal pancreas and chronic pancreatitis. We applied real-time PCR, immunohisto(cyto)chemistry, and nick-end labeling of apoptoses. In normal pancreas, a minor subset of acinar cells coexpressed TRAIL-R2 and TRAIL-R4, whereas ductular epithelium and interstitial fibroblast-like cells (FLC) expressed TRAIL-R4. TRAIL-R1 and TRAIL-R3 were not detected in normal pancreas. In chronic pancreatitis, the exocrine epithelium strongly expressed TRAIL-R1, -R2, -R4, and, to a lesser extent, TRAIL-R3. Islets focally neoexpressed TRAIL-R1 and -R2 and intensely expressed TRAIL-R4. Changes in TRAIL receptor expression were most pronounced in areas of inflammatory infiltration and active fibrosis. In normal pancreas, expression of TRAIL was low on the mRNA level and undetectable on the protein level. In chronic pancreatitis, FLC in areas of active fibrosis expressed TRAIL. In addition, apoptoses were most numerous in these areas. We show that these FLC are pancreatic stellate cells. Pancreatic stellate cells express TRAIL in vivo and in vitro, and TRAIL expression is enhanced by IFN-γ. Our findings indicate that the TRAIL/TRAIL receptor system is likely to be involved in chronic pancreatitis and suggest that pancreatic stellate cells may directly contribute to acinar regression by inducing apoptosis of parenchymal cells in a TRAIL-dependent manner.