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1
Tyrinova, Tamara, Olga Leplina, Sergey Mishinov, Marina Tikhonova, Evgeniya Dolgova, Anastasiya Proskurina, Vyacheslav Stupack, Sergey Bogachev, Alexander Ostanin, and Elena Chernykh. "Defective Regulation of Membrane TNFα Expression in Dendritic Cells of Glioblastoma Patients Leads to the Impairment of Cytotoxic Activity against Autologous Tumor Cells." International Journal of Molecular Sciences 21, no. 8 (April 21, 2020): 2898. http://dx.doi.org/10.3390/ijms21082898.
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Besides an antigen-presenting function and ability to induce antitumor immune responses, dendritic cells (DCs) possess a direct tumoricidal activity. We previously reported that monocyte-derived IFNα-induced DCs (IFN-DCs) of glioblastoma multiforme patients express low levels of membrane TNFα molecule (mTNFα) and have impaired TNFα/TNF-R1-mediated cytotoxicity against immortalized tumor cell line HEp-2. However, whether the observed defect could affect killer activity of glioma patient DCs against autologous tumor cells remained unclear. Here, we show that donor IFN-DCs possess cytotoxic activity against glioblastoma cell lines derived from a primary tumor culture. Granule-mediated and TNFα/TNF-R1-dependent pathways were established as the main mechanisms underlying cytotoxic activity of IFN-DCs. Glioblastoma patient IFN-DCs showed lower cytotoxicity against autologous glioblastoma cells sensitive to TNFα/TNFR1-mediated lysis, which was associated with low TNFα mRNA expression and high TACE/ADAM-17 enzyme activity. Recombinant IL-2 (rIL-2) and human double-stranded DNA (dsDNA) increased 1.5-fold cytotoxic activity of patient IFN-DCs against autologous glioblastoma cells. dsDNA, but not rIL-2, enhanced the expression of TNFα mRNA and decreased expression and activity of TACE/ADAM-17 enzyme. In addition, dsDNA and rIL-2 stimulated the expression of perforin and granzyme B (in the presence of dsDNA), suggesting the possibility of enhancing DC cytotoxicity against autologous glioblastoma cells via various mechanisms.
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Laurent, Guy, Fabien Guilloton, Aurelie de Thonel, Christine Jean, Cecile Demur, Veronique De Mas, and Anne Quillet-Mary. "TNFα Stimulates NKG2D-Mediated Lytic Activity of Acute Myeloid Leukemic Cells." Blood 106, no. 11 (November 16, 2005): 4411. http://dx.doi.org/10.1182/blood.v106.11.4411.4411.
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Abstract The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFα, they display cytolytic activity towards various cellular targets including CFU-GM. This mechanism is dependent on stimulation of Granzyme B/Perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patient with acute myeloid leukemia (AML) (FAB M0 to M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by pool >15% of leukemic cells. Futhermore, CD34+ hematopoietic progenitors undergoing granulo-monocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which upon stimulation by TNFα leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFα accumulation, such as AML or myelodisplasic syndromes.
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Chen, Chih-Chiun, Jennifer L. Young, Ricardo I. Monzon, Ningyu Chen, Viktor Todorović, and Lester F. Lau. "Cytotoxicity of TNFα is regulated by integrin-mediated matrix signaling." EMBO Journal 26, no. 5 (February 22, 2007): 1257–67. http://dx.doi.org/10.1038/sj.emboj.7601596.
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Miyaji, Chikako, Hisami Watanabe, Ryoko Miyakawa, Hisashi Yokoyama, Chika Tsukada, Yuiko Ishimoto, Shinobu Miyazawa, and Toru Abo. "Identification of effector cells for TNFα-mediated cytotoxicity against WEHI164S cells." Cellular Immunology 216, no. 1-2 (March 2002): 43–49. http://dx.doi.org/10.1016/s0008-8749(02)00525-7.
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Li, Y., T. D. Nguyen, A. C. Stechschulte, D. J. Stechschulte, and K. N. Dileepan. "Effect of mast cell granules on the gene expression of nitric oxide synthase and tumour necrosis factor-α in macrophages." Mediators of Inflammation 7, no. 5 (1998): 355–61. http://dx.doi.org/10.1080/09629359890884.
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Previous studies have shown that mast cell granules (MCG) inhibit numerous macrophage functions including tumour cytotoxicity, superoxide and nitric oxide (NO) production, and FCγ2a receptor-mediated phagocytosis. In this study, the effect of MCG on macrophage TNFα and nitric oxide synthase (iNOS) mRNA expression, and the production and fate of TNFα were examined. Upon activation with LPS+IFNγ, macrophages expressed both TNFα and iNOS mRNA and produced both TNFα and NO. Co-incubation of LPS+IFNγ-activated macrophages with MCG resulted in dose-dependent inhibition of iNOS mRNA expression. TNFα production in the activated macrophages was decreased by MCG, which was associated with a reduction in TNFα mRNA expression. MCG were also capable of degrading both macrophage-generated and recombinant TNFα. The direct effect of MCG on TNFα was partially reversed by a mixture of protease inhibitors. These results demonstrate that MCG decrease the production of NO and TNFα by inhibiting macrophage iNOS and TNFα gene expression. Furthermore, MCG post-transcriptionally alter TNFα levels via proteolytic degradation.
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Hu, Zhengbo, Ramya Viswanathan, Hui Cheng, Jianghong Chen, Xinping Yang, Angel Huynh, Paul Clavijo, et al. "Inhibiting WEE1 and IKK-RELA Crosstalk Overcomes TNFα Resistance in Head and Neck Cancers." Molecular Cancer Research 20, no. 6 (February 17, 2022): 867–82. http://dx.doi.org/10.1158/1541-7786.mcr-21-0624.
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Abstract TNFα is a key mediator of immune and radiotherapy-induced cytotoxicity, but many cancers, including head and neck squamous cell carcinomas (HNSCC), display TNF resistance due to activation of the canonical IKK–NF-κB/RELA pro-survival pathway. However, toxicities associated with direct targeting of the canonical pathway point to the need to identify mechanism(s) contributing to TNFα resistance and synthetic lethal targets to overcome such resistance in cancer cells. Here, RNAi screening for modulators of TNFα–NF-κB reporter activity and cell survival unexpectedly implicated the WEE1 and CDC2 G2–M checkpoint kinases. The IKKα/β-RELA and WEE1-CDC2 signaling pathways are activated by TNFα and form a complex in cell lines derived from both human papillomavirus (−) and (+) subtypes of HNSCC. WEE1 inhibitor AZD1775 reduced IKK/RELA phosphorylation and the expression of NF-κB–dependent pro-survival proteins Cyclin D1 and BCL2. Combination of TNFα and AZD1775 enhanced caspase-mediated apoptosis in vitro, and combination treatment with radiotherapy and AZD1775 potentiated inhibition of HNSCC tumor xenograft growth in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insight into the interplay between NF-κB signaling and WEE1-mediated regulation of the G2–M cell-cycle checkpoint in HNSCC. Implications: Inhibiting WEE1 and IKK-RELA crosstalk could potentially enhance the effects of therapies mediated by TNFα with less systemic immune suppression and toxicity than observed with direct interruption of IKK-NF-κB/RELA signaling.
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Almishri, Wagdi, Tania Santodomingo-Garzon, Tyson Le, Danuta Stack, Christopher H. Mody, and Mark G. Swain. "TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2." Journal of Innate Immunity 8, no. 6 (2016): 617–29. http://dx.doi.org/10.1159/000448077.
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NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses.
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Toni, Tiffany, Ramya Viswanathan, Yvette Robbins, Sreenivasulu Gunti, Xinping Yang, Angel Huynh, Hui Cheng, et al. "Combined Inhibition of IAPs and WEE1 Enhances TNFα- and Radiation-Induced Cell Death in Head and Neck Squamous Carcinoma." Cancers 15, no. 4 (February 6, 2023): 1029. http://dx.doi.org/10.3390/cancers15041029.
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Head and neck squamous cell carcinoma (HNSCC) remains a prevalent diagnosis with current treatment options that include radiotherapy and immune-mediated therapies, in which tumor necrosis factor-α (TNFα) is a key mediator of cytotoxicity. However, HNSCC and other cancers often display TNFα resistance due to activation of the canonical IKK–NFκB/RELA pathway, which is activated by, and induces expression of, cellular inhibitors of apoptosis proteins (cIAPs). Our previous studies have demonstrated that the IAP inhibitor birinapant sensitized HNSCC to TNFα-dependent cell death in vitro and radiotherapy in vivo. Furthermore, we recently demonstrated that the inhibition of the G2/M checkpoint kinase WEE1 also sensitized HNSCC cells to TNFα-dependent cell death, due to the inhibition of the pro-survival IKK-NFκB/RELA complex. Given these observations, we hypothesized that dual-antagonist therapy targeting both IAP and WEE1 proteins may have the potential to synergistically sensitize HNSCC to TNFα-dependent cell death. Using the IAP inhibitor birinapant and the WEE1 inhibitor AZD1775, we show that combination treatment reduced cell viability, proliferation and survival when compared with individual treatment. Furthermore, combination treatment enhanced the sensitivity of HNSCC cells to TNFα-induced cytotoxicity via the induction of apoptosis and DNA damage. Additionally, birinapant and AZD1775 combination treatment decreased cell proliferation and survival in combination with radiotherapy, a critical source of TNFα. These results support further investigation of IAP and WEE1 inhibitor combinations in preclinical and clinical studies in HNSCC.
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Ariana, Ardeshir, Norah A. Alturki, Stephanie Hajjar, Deborah J. Stumpo, Christopher Tiedje, Emad S. Alnemri, Matthias Gaestel, Perry J. Blackshear, and Subash Sad. "Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages." Journal of Biological Chemistry 295, no. 14 (February 24, 2020): 4661–72. http://dx.doi.org/10.1074/jbc.ra119.011633.
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The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88−/−, Trif−/−, MyD88−/−Trif−/−, MK2−/−, and Zfp36−/− mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)–induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.
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Souders, Christopher L., Juan J. Aristizabal-Henao, Sarah J. Patuel, John A. Bowden, Jasenka Zubcevic, and Christopher J. Martyniuk. "Interaction between Butyrate and Tumor Necrosis Factor α in Primary Rat Colonocytes." Biomolecules 13, no. 2 (January 30, 2023): 258. http://dx.doi.org/10.3390/biom13020258.
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Butyrate, a short-chain fatty acid, is utilized by the gut epithelium as energy and it improves the gut epithelial barrier. More recently, it has been associated with beneficial effects on immune and cardiovascular homeostasis. Conversely, tumor necrosis factor alpha (TNFα) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNFα are both linked with hypertension, studies have not yet addressed their interaction in the colon. Here, we investigated the capacity of butyrate to modulate a host of effects of TNFα in primary rodent colonic cells in vitro. We measured ATP levels, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial oxidative phosphorylation, and glycolytic activity in colonocytes following exposure to either butyrate or TNFα, or both. To address the potential mechanisms, transcripts related to oxidative stress, cell fate, and cell metabolism (Pdk1, Pdk2, Pdk4, Spr, Slc16a1, Slc16a3, Ppargc1a, Cs, Lgr5, Casp3, Tnfr2, Bax, Bcl2, Sod1, Sod2, and Cat) were measured, and untargeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) was employed to profile the metabolic responses of colonocytes following exposure to butyrate and TNFα. We found that both butyrate and TNFα lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNFα-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNFα-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNFα-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 (Pdk4) were increased via co-treatment of butyrate and TNFα, suggesting the synergistic inhibition of glycolysis. TNFα alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNFα on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNFα-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNFα and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNFα and butyrate in colonocytes that may be important in understanding conditions of the colon.
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Liu, Kebin, Najam ud Din, Darren D. Browing, Scott I. Abrams, and Dafeng Yang. "LTbR Mediates Cell Contact-Dependent CTL-Directed Anti-Tumor Cytotoxicity (50.11)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S92. http://dx.doi.org/10.4049/jimmunol.178.supp.50.11.
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Abstract Cytotoxic T lymphocytes (CTLs) are thought to use two cell contact-dependent effector mechanisms, perforin- and Fas-mediated cytotoxicity, to suppress tumor growth. Using a CTL adoptive immunotherapy model, we report here that perforin-deficient CTLs (pfp CTLs) exhibited a significant cytotoxicity against Fas-resistant tumors in vivo. We further demonstrated that the anti-tumor activity is directly mediated by the adoptively transferred CTLs. but does not involve CTL-secreted soluble molecules, such as IFNγ and TNFα as lytic agents, since these cytokines exhibited no direct cytotoxicity against these targets. Moreover, it was observed that LTβR, a cell-surface death receptor, is expressed on the tumor cell surface, and LTα, LTβ, and LIGHT, all of which are ligands for LTβR, were either constitutively expressed by or activated in the tumor-specific CTLs and primary CD8+ T cells. Silencing LTβR in these Fas-resistant tumor cells with LTβR-specific shRNA significantly decreased the ability of pfp CTL to mediate tumor rejection in vivo. Taken together, our data suggest that tumor-specific CTL use LTβR-mediated cytotoxicity as a third cell contact-dependent effector mechanism against tumor growth in vivo.
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Zhang, Li, El-Mustapha Haddouti, Hannes Beckert, Ralf Biehl, Shyam Pariyar, Julian M. Rüwald, Xian Li, et al. "Investigation of Cytotoxicity, Oxidative Stress, and Inflammatory Responses of Tantalum Nanoparticles in THP-1-Derived Macrophages." Mediators of Inflammation 2020 (December 3, 2020): 1–14. http://dx.doi.org/10.1155/2020/3824593.
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Tantalum (Ta) is gaining attention as a biomaterial in bone tissue engineering. Although the clinical advantage of Ta-based implants for primary and revision total joint replacement (TJA) has been well documented, few studies investigated the effect of wear products of Ta implants on peri-implant cells, and their potential contribution to aseptic implant loosening. This study is aimed at examining the cytotoxicity, oxidative stress, and proinflammatory potential of Ta and TiO2 nanoparticles (NPs) on macrophages in vitro. NPs were characterized using scanning electron microscopy, dynamic light scattering, and energy-dispersive X-ray. To test the NP-mediated cellular response in macrophages, THP-1-derived macrophages were challenged with both NPs, and cytotoxicity was analyzed using CCK-8 and LDH assays. Flow cytometry was used to investigate particle uptake and their internalization routes. NP-mediated oxidative stress was investigated by measuring the production of reactive oxygen species, and their proinflammatory potential was determined by quantifying the production of TNFα and IL-1β in cell culture supernatants using ELISA. We found that both Ta and TiO2 NPs were taken up through actin-dependent phagocytosis, although TiO2 NPs did also show some involvement of macropinocytosis and clathrin-mediated endocytosis. Ta NPs caused no apparent toxicity, while TiO2 NPs demonstrated significant cytotoxicity at a concentration of over 100μg/mL at 24 h. Ta NPs induced negligible ROS generation and proinflammatory cytokines (TNFα, IL-1β) in macrophages. In contrast, TiO2 NPs markedly induced these effects in a dose-dependent manner. Our findings indicate that Ta NPs are inert, nontoxic, and noninflammatory. Therefore, Ta could be considered an excellent biomaterial in primary and revision joint arthroplasty implants.
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Xie, Yanyun, Anthony Bruce, Lizhi He, Fengxiang Wei, Lijian Tao, and Damu Tang. "CYB5D2 enhances HeLa cells survival of etoposide-induced cytotoxicity." Biochemistry and Cell Biology 89, no. 3 (June 2011): 341–50. http://dx.doi.org/10.1139/o11-004.
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Cytochrome b5 domain containing 2 (CYB5D2) (neuferricin) belongs to the family of membrane-associated progesterone receptors (MAPRs). MAPRs affect multiple cellular processes, including proliferation, differentiation, and survival. Consistent with these observations, we report here that CYB5D2 enhances HeLa cells survival of etoposide (ETOP)-mediated cytotoxicity. Overexpression of CYB5D2 enhanced the survival of HeLa cells compared with HeLa cells transfected with empty vector (EV) upon ETOP treatment. As ETOP initiates ATM-dependent DNA damage response (DDR), we were able to show that CYB5D2 did not affect ETOP-induced DDR. In line with these observations, CYB5D2 did not protect HeLa cells from UV-induced cytotoxicity. Additionally, CYB5D2 had no effects on TNFα-induced apoptosis. Collectively, CYB5D2 enhances HeLa cell survival of ETOP-induced cytotoxicity with some specificity. CYB5D2 contains a cytochrome b5 (cyt-b5) domain and a transmembrane (TM) motif. Both domains are required for CYB5D2-mediated protection of HeLa cells from ETOP-induced cytotoxicity. In an effort to search for the underlying mechanisms, we have profiled gene expression between HeLa–CYB5D2 and HeLa–EV cells. Although ectopic CYB5D2 does not massively alter gene expression, the expression of several transcripts was affected more than 2-fold, suggesting that they may contribute to CYB5D2-mediated HeLa cell survival of ETOP treatment.
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Duffy, Joseph, Markella Zannikou, Rebecca Levine, Roger Stupp, David James, and Irina Balyasnikova. "IMMU-07. HUMANIZED BI-SPECIFIC ANTIBODY SHOWS POTENT ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii132. http://dx.doi.org/10.1093/neuonc/noac209.505.
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Abstract BACKGROUND Advancements in bioengineering have allowed bispecific Abs to develop with therapeutic applications in immuno-oncology. Here we describe a fully humanized bispecific t cell engager (BiTE) that binds the GBM-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2) and the T Cell receptor CD3 epsilon (CD3ε) to elicit an anti-tumor immune response in patient-derived models of GBM. METHODS In vitro, BiTE-mediated activation of T Cells was assessed via flow cytometry as measurements of T cell activation markers CD25 and CD69 and cytokines IFNγ and TNFα. BiTE-directed T cell-mediated cytotoxicity was assessed in GBM 6 and GBM12patient-derived xenograft lines. In vivo, GBM6 patient-derived xenograft cells were intracranially implanted in NSG-MHC I/II DKO mice, reconstituted with HLA-matched donor peripheral blood lymphocytes (PBMC), and subsequently treated with 2.5 mg/kg BiTE via i.p. injection x 4 days/week of therapeutic BiTE for three weeks. Animals were followed for survival, and the brains of long-term survivors were analyzed for residual disease and T cell infiltration. RESULTS In vitro, BiTE potently activated T cells as determined by a high expression of CD25 and CD69 and the production of TNFα and IFNγ. BiTE also directed T cell-mediated cytotoxicity against GBM cells in a dose-dependent manner, with significant activity still seen in the low nanogram range. In vivo, BiTE significantly extended the survival of tumor-bearing animals in GBM6patient-derived xenograft models. Histological analysis of long-term surviving mice shows complete absence of tumor and robust presence of CD8+ T cells localized to the original site of tumor implantation. CONCLUSIONS Our data demonstrate the efficacy of humanized BiTE in patient-derived GBM models, and our results support the investigation of this novel therapeutic in a clinical trial setting.FUNDING: R01 NS122395-01 and R01 NS095642-01
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Ma, Li, Haiyan Gong, Haiyan Zhu, Pei Su, Shannan Cao, Peng Liu, Jianfeng Yao, et al. "Identification Of a Novel Small-Molecule TNFα Inhibitor With Activity Against Inflammation In a Hepatitis Mouse Model." Blood 122, no. 21 (November 15, 2013): 4229. http://dx.doi.org/10.1182/blood.v122.21.4229.4229.
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Abstract Over-expression of tumor necrosis factor α (TNFα) is a hallmark of many inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease and septic shock and hepatitis, making it a potential therapeutic target for clinical interventions. To date, significant advances have been made in the development of biological agents targeting TNFα and its signaling components. There are several well known commercial TNFα inhibitors, such as infliximab, adalimumab and etanercept, all of which are TNFα antibodies or TNFR1-Fc chimeras and function to prevent TNFα from binding to its receptor. Those biomacromolecular agents have been proved to be effective in the treatment of inflammatory bowel disease and rheumatoid arthritis due to their unique superiorities such as high specificity. However, several severe limitations such as poor stability, cost-ineffective commercial-scale production and exclusion from blood/brain barrier have also emerged. Instead, small-molecule chemical compounds have been appreciated as appropriate alternatives for overcoming most disadvantages associated with macromolecular inhibitors. Furthermore, they offer additional clinical benefits such as simpler preparation for oral medicine. Now by the use of computer-aided drug design (CADD) and cell-based assays in vitro, several selective small-molecule antagonists of TNFα activity have been identified. They include broad-spectrum inhibitors targeting the key molecules of the intracellular TNFα pathway, functionally uncharacterized inhibitors of TNFα expression, inhibitors of the processing enzyme TNFα converting enzyme (TACE), and molecules that directly bind to TNFR or prevent TNFα-TNFR interactions. Although the small-molecule inhibitors are capable of blocking the biological activity of TNFα in vitro, few have been shown to abrogate or reduce TNFα-induced inflammatory responses in vivo and exhibit high IC50 and severe side effects. Also, none of the small-molecular inhibitors have been reported to successfully block TNFα’s interaction with TNFR through direct binding to TNFα. Thus, development of small molecules for TNFα therapy remains a major challenge. In this study, to explore chemical inhibitors against TNFα activity, we applied CADD combined with in vitro and cell-based assays and identified a lead chemical compound (named as C87 thereafter) from a compound library including about 90,000 small molecular compounds, which directly binds to TNFα indicated by SPR assay, and it potently inhibits TNFα-induced cytotoxicity (IC50=8.73μM) and effectively blocks TNFα–triggered signaling activities. More importantly, by using a murine acute hepatitis model, we showed that C87 attenuates TNFα-induced inflammation, thereby markedly reducing injuries to the liver and improving animal survival. Thus, our results lead to a novel and highly specific small-molecule TNFα inhibitor, which can be potentially used to treat TNFα mediated inflammatory diseases. Disclosures: No relevant conflicts of interest to declare.
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Krupka, Christina, Thomas Köhnke, Peter Kufer, Roman Kischel, Felix S. Lichtenegger, Franziska Brauneck, Stefan Rohling, Torben Altmann, Wolfgang Hiddemann, and Marion S. Subklewe. "Identifying Immune Resistance Mechanisms to CD33/CD3 BiTE® Antibody Construct (AMG 330) Mediated Cytotoxicity." Blood 126, no. 23 (December 3, 2015): 3677. http://dx.doi.org/10.1182/blood.v126.23.3677.3677.
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Abstract In our previous work, we showed elimination of primary acute myeloid leukemia (AML) cells by CD33/CD3 BiTE® antibody construct (AMG 330) mediated cytotoxicity (Krupka et al, Blood 2014). The goal of the present study was to identify innate and adaptive resistance mechanisms to AMG 330 mediated lysis of AML cells. Immune checkpoint molecules have been shown to be a highly relevant escape mechanism of malignant cells to evade innate and adaptive immunity. Previously, it was shown that AML cells upregulate the expression of inhibitory ligands in response to proinflammatory cytokines (Krönig et al, 2014). As AMG 330 mediated T-cell activation induces high levels of the proinflammatory cytokines IFNγ and TNFα, we assessed the constitutive and inducible expression profile of different immune checkpoint molecules on AML cell lines and primary AML cells, including PD-L1, HVEM, ILT3 and SLAMF7 by flow cytometry. No constitutive expression was observed for PD-L1 at time of primary diagnosis in 83.7% of the cases (103/123). In contrast, constitutive expression of HVEM and ILT3 was detected in 73.7% (42/57) and 91.9% (68/74) of patient samples, respectively. Adaptive resistance was evaluated by incubating AML cell lines and primary AML samples with IFNγ and TNFα. We observed an upregulation of PD-L1 and SLAMF7 on AML cell lines and on primary AML patient samples whereas HVEM and ILT3 did not show a significant change in expression level. To test the functional relevance of the immune checkpoint molecules upon AMG 330 mediated lysis, we used an ex vivo long term culture system that enabled us to analyse the dynamic process of receptor-ligand interaction over time. Blockade of the PD-1/PD-L1 interaction resulted in a significantly increase in AMG 330 mediated lysis of primary AML cells (n=9, p=0.03). Currently, blockade of the inducible molecule SLAMF7 in AMG 330 mediated cytotoxicity is being tested. Blocking of HVEM or ILT3 did not result in a significant increase in T cell activation and concomitant lysis of AML cells suggesting a less relevant role of HVEM and ILT3 in resistance to AMG 330 mediated cytotoxicity. The latter might also be influenced by the cytokine microenvironment which favours immune resistance of AML cells. Using a bead based multiplex assay we screened the bone marrow (BM) plasma from 16 AML patients and 3 healthy donors (HD) for the presence of 33 cytokines. The cytokine profile differed between AML patients and healthy donors (HDs). The plasma levels of IL-8, IP-10 and CXCL-16 were higher in the AML samples compared to those of HDs (p=0.0041, 0.0248 and 0.0289, respectively). In contrast, EGF, FLT3-ligand, RANTES and IL-4 were significantly lower in AML samples compared to HDs (p=0.0227, 0.0145, 0.0041 and 0.0041, respectively). However, we did observe a high inter-patient variability of cytokine composition in AML. To explore the functional relevance of the BM plasma on AMG 330 mediated cytotoxicity, cocultures of AML cell lines and HD T cells were set up using different sources of plasma including fetal calf serum (FCS) and patient derived BM plasma. Interestingly, AMG 330 mediated cytotoxicity was significantly reduced using patient derived BM plasma (n=5) compared to cultures containing FCS (n=4) (mean % lysis FCS 97.4 vs PT 70.6). This was accompanied by a considerable impairment in T-cell proliferation (mean % proliferation FKS 44.7% vs PT 26.6%). Currently, we are investigating which soluble factors are responsible for the immunosuppressive effects and if we can increase lysis efficacy and T-cell proliferation through specific blocking of them. In summary we have identified possible resistance mechanisms of AML cells to AMG 330 mediated cytotoxicity. Dynamic receptor-ligand interactions between target and effector cells as well as soluble factors contribute to AMG 330 mediated lysis of primary AML cells. We hypothesize that AMG 330 mediated cytotoxicity can be augmented through combinatorial approaches including PD-1 blockade. The significance of our findings will first be validated in an in vivo mouse model and prospectively translated into human studies. Disclosures Krupka: AMGEN Research (Munich): Research Funding. Kufer:AMGEN Research (Munich): Employment, Equity Ownership. Kischel:AMGEN Research (Munich): Employment, Equity Ownership. Subklewe:AMGEN Research (Munich): Research Funding.
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Crocetti, Jillian, Oscar Silva, Lisa Humphries, and M. Miceli. "The role of Discs Large Homolog 1 phosphorylation in translating TCR signal strength into discrete CTL functions (P1168)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 190.9. http://dx.doi.org/10.4049/jimmunol.190.supp.190.9.
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Abstract In cytotoxic T lymphocytes (CTLs), engagement of the T cell receptor (TCR) triggers signaling pathways that culminate in cytokine production and cytotoxic killing. TCR signal strength is linked to CTL function with low signal triggering cytotoxicity while high signal leads to both cytotoxicity and cytokine production. Scaffold protein Dlg1 (Discs Large Homolog 1) is a key point of control downstream of the TCR that coordinates both cytokine production and cytotoxic functional output. However, the molecular mechanism by which Dlg1 regulates cytokine production and cytotoxicity in response to TCR signal strength remains elusive. We report that Dlgh1 is tyrosine phosphorylated in response to TCR stimulation, and that phosphorylation is a key step in Dlg1-mediated activation of the alternative p38 pathway and downstream cytokine gene expression. We demonstrate that Dlg1 phosphorylation is mediated by Dlg1-associated Lck, not Zap70; and that Dlg1 Tyr222 is a major site of TCR-induced phosphorylation. Importantly, T cells expressing Dlg1 with a tyrosine to phenylalanine mutation (Y222F) show a defect in TCR-induced activation of the alternative p38 pathway, activation of NFAT and transcriptional upregulation of TNFα and IFNγ genes, but not the transcriptional upregulation of the granzyme B gene or CTL degranulation. These data position Dlg1 phosphorylation as a molecular switch allowing selective activation of p38-dependent events in response to the strength of the TCR signal.
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Partheniou, Fotini, Stephen M. Kelsey, Srinivasa M. Srinivasula, Adrian C. Newland, Emad S. Alnemri, and Li Jia. "c-IAP1 Blocks TNFα-Mediated Cytotoxicity Upstream of Caspase-Dependent and -Independent Mitochondrial Events in Human Leukemic Cells." Biochemical and Biophysical Research Communications 287, no. 1 (September 2001): 181–89. http://dx.doi.org/10.1006/bbrc.2001.5582.
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Kuhla, Angela, Christian Eipel, Nikolai Siebert, Kerstin Abshagen, Michael D. Menger, and Brigitte Vollmar. "Hepatocellular apoptosis is mediated by TNFα-dependent Fas/FasLigand cytotoxicity in a murine model of acute liver failure." Apoptosis 13, no. 12 (October 23, 2008): 1427–38. http://dx.doi.org/10.1007/s10495-008-0269-7.
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Gong, Haiyan, Yigeng Cao, Peng Liu, Haiyan Zhu, Zhezhe Wang, Fei Xiang, Tong Wang, et al. "Successful Treatment of Animal Models of Acute Graft-Versus-Host Disease with Small-Molecule TNF Inhibitor." Blood 128, no. 22 (December 2, 2016): 4714. http://dx.doi.org/10.1182/blood.v128.22.4714.4714.
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Abstract Tumor necrosis factors(TNF, including TNFα and TNFβ) have now been shown to be two of the major mediators of inflammation and can induce other inflammatory cytokines and proteases that orchestrate inflammatory responses. Recently, many reports have indicated that overexpression of TNF involved in the development of acute graft-versus-host disease(aGVHD), and TNF inhibitors have tremendous potential for the treatment of this disease. To date, 5 of the current biomacromolecular TNF inhibitors, including infliximab (IFX), etanercept(ETN), adalimumab, golimumab, and certolizumab pegol, are available for the part of the routine treatment of patients with rheumatoid arthritis, Crohn's disease, aGVHD and other inflammatory diseases. However, over the past years, several severe limitations of those biomacromolecular TNF inhibitors have also emerged, such as poor stability, exclusion from blood/brain barrier and inducing the development of antidrug antibodies (ADA). For this reason, small-molecule chemical compounds have been designed and developed as appropriate alternatives for overcoming those limitations associated with biomacromolecular inhibitors. We previously reported a small molecular TNFα inhibitor C87, a lead chemical compound composed of (E)-4-(2-(4-chloro-3-nitrophenyl) which could directly binds to TNF-α, was designed by computer-aided drug design(CADD) and identified by virtual screening and cell-based assays in vitro, and it could attenuated mouse acute hepatitis mediated by TNFα and improve the mouse survival in vivo. As the docking template for virtual screening of above was based on the crystal structure of TNFβ·TNFR1 complex, C87 was supposed to has the potential to be a small molecule inhibitor of TNFβ which few reports to date have indicated . Therefore, in this study, the inhibition of C87 on TNFβ-induced cytotoxicity will be elucidated in detail in vitro, and more importantly, whether C87 could effectively blocked the development of aGVHD in animal model will also be further investigated. To address those issues, we measured the inhibitory activity of C87 on TNF-mediated cytotoxicity in vitro or in vivo, the results indicated that C87, dissolved in a optimal clinical solvent, could potently and specifically inhibit the TNFβ-induced cytotoxicity in L929 cells in a dose-dependent fashion(IC50=1.59μM). C87 could effectively block TNFβ-induced the activation of Caspase3 and Caspase8 in L929 cells( cultured for 24 hours) and maintain the rate of cell survival up to 80%. In addition, C87 could also suppress the up-regulation of IL-1β and TNFβ itself induced by TNFβ, which was comparable with the TNFβ-neutralizing antibody treatment. Strikingly, C87 could directly bind to TNFβ with high affinity indicated by SPR assay(KD=750 nM). Most importantly, by using a acute GVHD mouse model, low dose C87 (2mg/kg), as single drug treatment for GVHD, could prevent the development of aGVHD in experimental mice efficiently by intraperitoneal administered. The survival rate of low dose C87 groups were 100%(control, 20%), and Low dose C87 also significantly attenuated the aGVHD severity in the damaged organs (such as skin and liver) by using histopathological analysis. And the results of further identification of inflammatory factors in the serum of the treated mice, comparing to the control mice, indicated that more than five kind of cytokines, including IL-2, IL-6, IL-9, IL-18 and GRO-α, were markedly decreased, similar potency to the CSA. Taken together, we demonstrated that small molecular inhibitor C87 could inhibit both TNFα and TNFβ-induced cytotoxicity and effectively block TNF-triggered signaling activation. Importantly, in addition to its efficacy in vitro, C87 showed the exciting treatment effect in aGVHD mouse model at low dose. Thus, our results extended previous work and suggested that C87 is one of the first effective small-molecule inhibitors of TNF identified to date in the treatment for aGVHD in animal model and has the potential to be a novel small molecular agent in treatment for TNF- mediated inflammatory disease in the future. Disclosures No relevant conflicts of interest to declare.
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Silbermann, Rebecca, Dan Zhou, Jumpei Teramachi, Xiang-Qun Xie, G. David Roodman, and Noriyoshi Kurihara. "The p62-ZZ Domain Inhibitor XRK3F2 Alters Myeloma-Induced Suppression of Osteoblast Differentiation and Is Highly Cytotoxic to Myeloma Cells in Combination with Bortezomib." Blood 124, no. 21 (December 6, 2014): 2083. http://dx.doi.org/10.1182/blood.v124.21.2083.2083.
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Abstract Multiple myeloma (MM) bone disease results in increased osteoclastic bone destruction and irreversible osteoblast (OB) suppression. Cell-cell interactions between MM cells and bone marrow stromal cells (BMSC), which are OB precursors, activate signaling pathways in BMSC that result in enhanced MM cell growth, osteoclast formation, and inhibition of OB differentiation. OB inhibition results from suppression of key OB transcription factors, such as RUNX2, and induction of Gfi1, a transcriptional repressor of RUNX2. We reported that the ZZ domain of p62 (sequestosome-1) is required for BMSC support of MM cell growth and acts as a signaling hub for the formation of BMSC signaling complexes activated by MM cells and TNFα, resulting in NFκB, p38MAPK, and PI3K activation. We developed a small molecule inhibitor targeting the ZZ domain, XRK3F2 (previously CMPD3) that blocks p62-ZZ domain-mediated protein interactions, including TNFα-mediated signaling in BMSC. Further, we found that XRK3F2 was directly cytoxic to MM cells at high concentrations (5TGM1 murine MM cell IC50 4.35μM). Recently, we reported that administration of XRK3F2 two weeks after intratibial injection of 5TGM1 MM cells induces dramatic new cortical bone formation adjacent to MM cells in an immunocompetent in vivo model of MM bone disease. Additionally, bones from XRK3F2 treated animals not inoculated with tumor did not demonstrate new bone formation. This suggests that XRK3F2 changes the local effect of MM on bone at concentrations that were not sufficient for MM cell death. We now report that XRK3F2 alters MM-induced suppression of OB differentiation, and that the combination of XRK3F2 and bortezomib increases in vitroMM cell cytotoxicity. To determine if the dramatic bone formation induced by XRK3F2 resulted from reduced production of OB inhibitors by MM cells, enhanced BMSC differentiation to OB, or alteration of MM cell-BMSC interactions, we treated 5TGM1 cells, MC4 (murine stromal) cells, and 5TGM1-MC4 co-cultures with XRK3F2 and evaluated expression of RUNX2, Gfi1, and other key OB differentiation genes by qPCR. XRK3F2 treatment of 5TGM1 cells did not change 5TGM1 production of the OB inhibitors IL-7 or TNFα, and did not alter osteogenic differentiation of MC4 cells. However, XRK3F2 treatment of 5TGM1-MC4 co-cultures performed with a transwell insert increased MC4-derived RUNX2 expression 3-fold, as compared with vehicle treated co-cultures, and fully blocked the induction of Gfi1 in MC4 cells by MM cells (MC4-5TGM1 co-culture resulted in 12.4-fold increase in MC4 cell Gfi1 expression as compared with MC4 expression of Gfi1 without MM cell co-culture.) XRK3F2 treatment of MC4 cells also blocked TNFα-induced upregulation of Gfi1 (TNFα treatment increased MC4 cell Gfi1 expression 16.4-fold compared with control.) These results suggest that XRK3F2 blocks the TNFα-induced suppression of OB differentiation observed in MM cell-BMSC co-cultures through it’s effects on TNFα-mediated signaling facilitated by the p62-ZZ domain. To determine if we could further enhance the effects of XRK3F2 on bone formation and tumor burden, we evaluated if bortezomib, an anti-MM drug with potential bone anabolic effects, enhanced XRK3F2-induced MM cell cytotoxicity. Human MM cell lines were treated with combinations of XRK3F2 and bortezomib at concentrations below the IC50 of each drug for each cell line. H929, MM1.S, U266, ANBL6, and RPMI8226 cells were treated for 48 hours with XRK3F2, bortezomib (1nM), or the combination, and analyzed by MTT assay for viability. All cell lines demonstrated a highly significant decrease in viability in response to treatment with the XRK3F2-bortezomib combination as compared with either alone (p < 0.01). These results suggest that XRK3F2 or its derivatives, in combination with bortezomib or other proteasome inhibitors, should have a profound therapeutic effect on MM tumor burden and bone disease. Disclosures Silbermann: Amgen: Consultancy; Celgene: Research Funding. Roodman:Eli Lilly and Co.: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.
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Silva, Oscar, Jillian Crocetti, Lisa Humphries, and M. Miceli. "DLG1 alternative splice variants: molecular conduits specifying proximal TCR signals to distinct CTL functional responses (P1174)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 190.12. http://dx.doi.org/10.4049/jimmunol.190.supp.190.12.
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Abstract CD8+ cytotoxic T lymphocytes (CTLs) are a critical component of the adaptive immune system because they mediate contact-dependent cytolysis and produce proinflammatory cytokines. Although both of these CTL activities require T cell receptor (TCR) activation these functions are not always coordinately invoked, suggesting that signaling pathways downstream of the TCR can be selectively activated to guide specific functional outcomes. Here we investigated the role of Discs large homolog 1 (DLG1) alternative splice variants, DLG1 i1A-i1B and DLG1 i1B, in coupling proximal TCR signals to select downstream pathways to specify cytotoxicity and proinflammatory cytokine transcription in CD8+ T cells. We found that DLG1 i1A-i1B, but not DLG1 i1B selectively directs TCR-induced p38/NFAT-dependent proinflammatory cytokine production by binding Lck, promoting alternative p38 activation and downstream NFAT mediated upregultion of IFNγ and TNFα gene expression. Conversely, DLG1 i1A-i1B mediated alternative p38 activation had no effect on actin polymerization, granzyme B gene expression, granzyme B release, or degranulation. Rather, either DLG1 variant expressed in CD8+ T cells promoted antigen-induced degranulation, suggesting that both DLG1 variants have p38-independent activity guiding granule-dependent cytotoxicity. Thus CD8+ T cells contain two distinct DLG1 scaffolds that act as molecular conduits for proximal TCR signals to allow differential CTL functional responses.
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Bissonnette, E. Y., R. Mathison, L. Carter, J. S. Davison, and A. D. Befus. "Decentralization of the Superior Cervical Ganglia Inhibits Mast Cell Mediated TNFα-Dependent Cytotoxicity.1. Potential Role of Salivary Glands." Brain, Behavior, and Immunity 7, no. 4 (December 1993): 293–300. http://dx.doi.org/10.1006/brbi.1993.1029.
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de Weerdt, Iris, Sanne Terpstra, Tom Hofland, Roeland Lameris, Renee C. G. de Bruin, Mark-David Levin, Ester B. M. Remmerswaal, et al. "Chronic Lymphocytic Leukemia (CLL) Cells Are Susceptible to γδ-T Cell Mediated Killing, Provided CLL-Derived γδ-T Cell Dysfunction Can be Reversed." Blood 126, no. 23 (December 3, 2015): 2914. http://dx.doi.org/10.1182/blood.v126.23.2914.2914.
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Abstract Background: Although T cell immunotherapy is considered a promising therapeutic approach in B cell malignancies, autologous T cell based therapy proved to be far less effective in CLL than in more aggressive B cell malignancies. This has been attributed to an acquired state of T cell dysfunction. Disturbances in conventional (αβ-)T cells include expansion of CD4+ and CD8+ T cells, increased expression of exhaustion markers and impaired cytotoxicity and cytokine production. Vγ9Vδ2-T cells are a conserved subset of cytotoxic T lymphocytes with potent antitumor activity, due to recognition of phosphoantigen-induced changes in CD277 in tumor cells. Aminobisphosphonate (ABP) treatment leads to intracellular accumulation of phosphoantigens and increased Vγ9Vδ2 antitumor responses. Vγ9Vδ2-T cells have been shown to effectively kill malignant B cell lines in vitro. Moreover, in clinical trials Vγ9Vδ2-T cells have been shown to recognize and kill B cell lymphomas. Whether Vγ9Vδ2-T cells could be exploited for CLL immunotherapy has not yet been explored. The aim of this study is to investigate the phenotype and function of Vγ9Vδ2-T cells in CLL patients, in order to determine whether Vγ9Vδ2-T cells can effectively kill CLL cells. Results: Frequencies of Vγ9Vδ2-T cells do not differ between untreated CLL patients (n=46) and age-matched healthy controls (HC) (n=20) as assessed by flow cytometry. Vγ9Vδ2-T cell subpopulations are skewed towards effector type (CD27- CD45RA-) in CLL patients, while numbers of naïve (CD27+ CD45RA+) Vγ9Vδ2-T cells are decreased. Expression of exhaustion markers PD-1 and BTLA is comparable between CLL and HC, as is expression of CD16, mediating antibody-dependent cellular cytotoxicity. Next, we compared the functionality of Vγ9Vδ2-T cells from CLL patients and HC. We first examined cytokine production and CD107a expression, a marker of degranulation. Production of TNFα and IFNγ upon PMA/ionomycin stimulation was significantly diminished in CLL Vγ9Vδ2-T cells as compared to HC Vγ9Vδ2-T cells. Similarly, CD107a expression was significantly reduced. Overnight coculture with primary CLL cells or the Vγ9Vδ2-T cell sensitive Daudi lymphoma cell line also induced expression of TNFα, IFNγ and CD107a. However, upon co-culture, HC Vγ9Vδ2-T cells expressed significantly more TNFα, IFNγ and CD107 than CLL Vγ9Vδ2-T cells. Subsequently, we compared cytotoxicity of Vγ9Vδ2-T cells towards Daudi cells. HC-derived Vγ9Vδ2-T cells killed Daudi cells 3-4 times more effectively at 1:5 and 1:2.5 effector:target ratios. Although ABP pretreatment of Daudi cells increased both CLL-derived and HC-derived Vγ9Vδ2-mediated killing, differences between CLL and HC could not be overcome. We then looked at Vγ9Vδ2-T cell cytotoxicity towards CLL cells. Vγ9Vδ2-T cells from HCs effectively recognized and killed primary CLL cells, irrespective of ABP pretreatment. CLL-derived Vγ9Vδ2-T cells killed allogeneic CLL cells significantly less efficiently. Finally, we investigated whether the Vγ9Vδ2-T cell dysfunction in CLL patients was reversible upon ex vivo activation without the presence of leukemic B cells. Purified Vγ9Vδ2-T cells were cocultured with mature monocytic-derived dendritic cells in the presence of ABP for 8 days. Following these culture conditions, no difference was observed in production of TNFα, IFNγ and IL-4 upon PMA/ionomycin stimulation between HC- and CLL-derived activated Vγ9Vδ2-T cells. Likewise, there was no difference in CD107a expression. The activated Vγ9Vδ2-T cells of HCs and CLL patients were equally effective at killing Daudi cells. Conclusion: Vγ9Vδ2-T cells are capable of recognizing and killing CLL cells. Yet, CLL-derived Vγ9Vδ2-T cells are functionally impaired in terms of cytokine production and cytotoxic capacity in comparison to age-matched HCs. Functional impairments of Vγ9Vδ2-T cells are reversible upon ex vivo activation. If dysfunction can be overcome effectively, the antileukemic properties of autologous Vγ9Vδ2-T cells can be efficiently employed. Disclosures No relevant conflicts of interest to declare.
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Kashyap, Trinayan, Boris Klebanov, Christian Argueta, Margaret S. Lee, Sharon Shacham, Michael Kauffman, Yosef Landesman, and William Senapedis. "Efficacy of Selinexor Is Dependent on IκB-α Expression and NF-Kb Deactivation in Multiple Myeloma Cells." Blood 128, no. 22 (December 2, 2016): 5660. http://dx.doi.org/10.1182/blood.v128.22.5660.5660.
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Abstract Background: Selinexor is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) including p53, pRB, and IκB-α. Selinexor has therapeutic benefit both pre-clinically and clinically (NCT01607892, NCT02336815) in multiple myeloma (MM). It has been previously demonstrated that the extent of NF-κB transcriptional inhibition is one of the critical mechanisms contributing to the efficacy and/or resistance to selinexor in cells. However, the mechanism leading to NF-κB inhibition after selinexor treatment is not fully understood. We hypothesized that the level of the cellular inhibitor of NF-κB, IκB-α, and its compartmental localization play an important role in NF-κB transactivation and response to selinexor. In this study, we investigate the effect of selinexor treatment on MM cells with low expression of IκB-α and high NF-kB activity in order to understand the mechanism of NF-κB inhibition by selinexor. Methods: IM9 and RPMI-8226cells were treated with selinexor in the presence or absence of 20 ng/mL tumor necrosis factor α (TNFα; inflammatory mimic) and whole protein lysates were analyzed by immunoblotting. Cytotoxic effects of selinexor were evaluated using standard viability assays. IκB-α knockdown was performed using transfection of specific siRNA duplexes. NF-κB transcriptional activity was analyzed using an ELISA assay. Results: Selinexor induces nuclear localization of IκB-α in MM cells. RNAi of IκB-α in MM cells reduced the cytotoxic effects of selinexor by 10-fold. In addition, knockdown of IκB-α reduces the synergy of the selinexor plus proteasome inhibitor (bortezomib or carfilzomib) combination. This data suggests that in MM, IκB-α plays a major role in cellular sensitivity to selinexor potentially through NF-κB activity. Selinexor inhibited NF-kB transcriptional activity in IM9 and RPMI-8226 cells with IC50 of 1079 nM and 591 nM respectively. Although the difference in NF-κB activity IC50 is only 2-fold between the two MM cell lines (MTT IC50s are ~100 nM), IM9 cells have a 100-fold higher basal NF-kB activity when compared to RPMI-8226 cells. Under TNFα stimulation NF-kB activity was induced by 1.5- and 35-fold in IM9 and RPMI-8226, respectively. We observed that selinexor treatment caused a dose dependent inhibition of IκB kinase (IKK)-mediated phosphorylation of serine 32/36 on IκB-α and serine 536 on the NF-κB p65 subunit (RelA) upon TNFα stimulation in both cell lines. In RPMI-8226 cells, selinexor reduced TNFα-induced IκB-α phosphorylation in a dose dependent manner and protected IκB-α from degradation. In IM9 cells that have high basal NF-κB activity, TNFα did not induce NF-kB activity or cause IκB-α degradation. However, selinexor treatment inhibited NF-kB activity below its basal level (70% reduction) which resulted in dose dependent reduction in the level of IκB-α protein perhaps through inhibition of NF-κB transcriptional control of IκB-α mRNA expression. Conclusions: IκB-α plays a major role in the cellular cytotoxicity of selinexor in cancer cells. Multiple myeloma cells lose sensitivity to selinexor treatment upon IκB-α silencing, which in turn reduces the cytotoxicity of selinexor. TNFα stimulation induces the phosphorylation of NF-κB p65 subunit and IκB-α through increased IKK activity resulting in IκB-α degradation and NF-κB activation. In RPMI-8226 cells, selinexor treatment blocked TNFα-induced degradation of IκB-α. However, in IM9 cells TNFα alone did not have any significant effect on IκB-α which might be due to the high basal NF-kB activity. Interestingly, IκB-α is also a transcriptional target of NF-kB. In IM9 cells, selinexor treatment reduces NF-kB activity below the high basal level in a dose dependent manner resulting in near complete inhibition of NF-kB-controlled IκB-α mRNA transcription and a loss of the IκB-α protein. Ultimately, selinexor treatment inhibits cell viability and NF-kB transcriptional activity regardless of basal NF-κB activity in MM cells. Because of this IκB-α/NF-kB transcriptional mechanism, selinexor treatment can inhibit both chronic (unresponsive to TNFα) and acute (TNFα-simulated) inflammatory signaling which makes selinexor an applicable therapy to cancer cells with a variety of aberrant signaling pathways. Disclosures Kashyap: Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta:Karyopharm Therapeutics: Employment, Equity Ownership. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership.
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Hideshima, Teru, Pierfrancesco Tassone, Dharminder Chauhan, Kenji Ishitsuka, Constantine Mitsiades, Noopur Raje, Shaji Kumar, et al. "Targeting IKK Inhibits Multiple Myeloma (MM) Cell Growth in the Bone Marrow Microenvironment." Blood 104, no. 11 (November 16, 2004): 2351. http://dx.doi.org/10.1182/blood.v104.11.2351.2351.
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Abstract NF-κB is a transcriptional factor promoting tumor cell growth and inhibition of apoptosis via regulating expression of proteins modulating cell cycle and anti-apoptosis. NF-κB is constitutively associated with an inhibitor (IκB), which is phosphorylated by IκB kinase (IKK) upon cell stimulation (ie, TNFα) and subsequently degraded by ubiquitin-proteasome pathway, thereby allowing NF-κB to translocate to nucleus. We have previously shown that an IKK inhibitor PS-1145 partially (20–50%) inhibits MM cell proliferation; however, it inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. Targeting IKKβ is therefore a possible therapeutic option for inhibition of MM cell growth in the bone marrow microenvironment by downregulating NF-κB activity. In this study, we further delineated the biologic significance of IKK inhibition in MM cells using IKKβ specific inhibitor MLN120B (Millennium Pharmaceuticals, Cambridge, MA). MLN120B induced 60–90% growth inhibition in cells from the MM cell lines RPMI8226, RPMI/Dox40, RPMI/LR5, U266, and INA-6; on the other hand, it induced only 25–30% inhibition in MM.1S and MM.1R cells, assessed by 72 h tritiated-thymidine uptake. Importantly, neither IL-6 nor IGF-1 overcomes the growth inhibitory effect of MLN120B in both MM.1S and U266 cells. Interestingly, MLN120B significantly augmented TNFα-induced cytotoxicity in MM.1S cells. We next examined whether MLN120B could enhance the cytotoxicity of other agents. MLN120B augmented growth inhibition triggered by doxorubicin and melphalan in RPMI8226 and IL-6 dependent INA-6 cell line. We therefore studied growth inhibitory effect of MLN120B in the presence of bone marrow stromal cells (BMSCs). MLN120B inhibited 70–80% of constitutive IL-6 secretion from BMSCs, without toxicity. Importantly, MLN120B almost completely blocked stimulation of MM.1S, U266 and INA-4 cell growth and IL-6 secretion from BMSCs induced by binding of tumor cells to BMSCs. Finally, MLN120B overcame the protective effect of BMSCs, cell adhesion mediated drug resistance, against dexamethasone in MM.1S cells. Taken together, our data demonstrate that an IKKβ inhibitor induces cytotoxicity in MM cells in the BM milieu, providing the framework for clinical trials of these novel agents to improve patient outcome in MM.
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Hudig, Dorothy, Christopher McKinney, Bryce Alves, David Tamang, Doug Redelman, Cherie Singer, and Viki Elliott. "Cytotoxic mechanisms of IL-4 polarized CTLs (101.28)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 101.28. http://dx.doi.org/10.4049/jimmunol.184.supp.101.28.
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Abstract IL-4 down-regulates perforin and granzymes and depresses rapid (4 hr) cytotoxic T lymphocyte (CTL) killing. Curiously, IL-4 can increase anti-tumor immunity in animal models. We wanted to know how IL-4 polarized CTLs kill and found that they have potent delayed cytotoxicity. We cultured BALB/c mouse splenocytes with conA and 500 units/ml mouse IL-4 or IL-2 for 3 days and then re-cultured the cells for 3 days with cytokines without conA. The IL-4 CTLs were polarized as indicated by expression of the IL-4 inducible lipase PLRP2. By flow cytometry, IL-4 CTLs expressed Grz B at lower levels than IL-2 CTLs. We assayed cytotoxicity using anti-CD3-redirected death of P815 tumor cells. Death was measured at 48 hrs by loss of EGFP+ P815 cells using flow cytometry with cell count beads to account for dead cell disintegration. Cytotoxicity by the IL-4 CTLs was 80% at effector lymphocyte to P815 (E:T) ratios of 1:10. IL-4 CTLs had perforin-independent mechanisms, with similar activity for WT and Prf1-/- CTLs. We monitored T cells for death receptor ligands. Quantitative RT-PCR indicated that IL-4 cells maintained comparable levels of mRNA for LTβ, LIGHT, TRAIL and FasL, but had lower levels of TNFα and TNFβ, than IL-2 CTLs. We conclude that IL-4 polarized CTLs have potent cytotoxicity which could be mediated by death receptor ligands and/or by novel IL-4 inducible potential cytotoxins. Supported in part by NIH grants R01CA38942, T32CA09563, P20 RR-016464 & P20-RR015581.
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Baldea, Ioana, Sorina Danescu, Flaviu Tabaran, Adriana Gabriela Filip, Rodica Mariana Ion, Diana Elena Olteanu, Alexandra Cristina Sevastre-Berghian, et al. "Inhibition of Survival Mechanisms and Cell Death Induction in Melanoma Following Photodynamic Therapy Mediated by Meso-5,10,15,20-tetrakis-(4-hydroxyphenyl)-porphyrin." Processes 11, no. 3 (March 17, 2023): 917. http://dx.doi.org/10.3390/pr11030917.
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(1) Background: Photodynamic therapy (PDT) involves the selective killing of tumor cells by the generation of reactive oxygen species using a photosensitizer (PS) activated by irradiation. In melanoma, PDT efficiency is altered by several mechanisms, such as the presence of melanin and melanosomes and pro-survival pathways mediated by transcription factors such as: AP-1 (activator protein), MITF (microphthalmia inducible transcription factor), HIF1α (hypoxia inducible factor), and NF-kB (nuclear factor kappa B). The study aimed to investigate the anti-melanoma effects of PDT mediated by meso-5,10,15,20-tetrakis-(4-hydroxyphenyl)-porphyrin (THPP) as a photosensitizer. (2) Methods: Cocultures of melanoma, two human, WM35 and M1–15, and murine B16-F10, with endothelial cells, were used. Cytotoxicity, oxidative damage, angiogenesis markers, and melanogenesis were assessed using colorimetry, flowcytometry, confocal microscopy, spectrophotometry, ELISA, and Western blotting. (3) Results: The maximal killing efficiency of PDT was reached in WM35, followed by M1–15, and then B16-F10, and it occurred through both apoptosis and necrosis. Although constitutive pigmentation diminished the PDT efficiency, de novo melanogenesis exhibited no protection. PDT increased TNFα, and inhibited NFkB, MITF, HIF1α, and AP1, leading to inflammation and angiogenesis markers’ inhibition. (4) Conclusions: THPP-mediated PDT efficiently induced cell death through apoptosis, necrosis, and the inhibition of pro-survival pathways mediated by NFkB, AP1, HIF1α, and MITF in the melanoma coculture models.
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Yin, Yuzhi, Paul D. Allen, Li Jia, Stephen M. Kelsey, and Adrian C. Newland. "8-Cl-adenosine mediated cytotoxicity and sensitization of T-lymphoblastic leukemia cells to TNFα-induced apoptosis is via inactivation of NF-κB." Leukemia Research 25, no. 5 (May 2001): 423–31. http://dx.doi.org/10.1016/s0145-2126(00)00147-8.
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Norkina, Oxana, Archana Thakur, Maxim Norkin, Elyse Paul, Zaid Al-Kadhimi, and Lawrence G. Lum. "Activated T Cells (ATC) Armed with Anti-CD3 X Anti-Her2/Neu Bispecific Antibody (Her2Bi) Is a Potent Immunomodulator." Blood 112, no. 11 (November 16, 2008): 2557. http://dx.doi.org/10.1182/blood.v112.11.2557.2557.
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Abstract In our ongoing phase I dose-escalation trial in women with metastatic breast cancer, multiple infusions of ATC armed with Her2Bi (aATC) induced elevated levels of IL-2, IFNγ TNFα, GM-CSF, and IL-12 as well as the development of cytotoxic activity directed at breast cancer cell lines lasting up to 4 months (Grabert et al. Clin Cancer Res. 2006). These results suggested that aATC infusions “vaccinated” the endogenous immune system of the patients. In this study, we explored mechanisms of the increased cytotoxicity after immunotherapy with armed ATC by testing for cell-mediated cytotoxicity and in vitro anti-tumor antibody synthesis. We co-cultured irradiated aATC with fresh PBMC in presence or absence of SKBR3 for 7 days, collected the PBMC, and tested for cytotoxicity in MTT and Cr51 release assays directed at SKBR3, Daudi and A-431 cell lines. PBMC mediated high levels of non-specific cytotoxicity against all tested cell lines and there were no phenotypic changes in the PBMC after 7 days of co-culture. When PBMC were co-cultured with irradiated aATC and SKBR3 for 21 days in presence of IL-2, the B cells in the PBMC produced significantly higher amounts of specific Abs directed SKBR3 (ELISA for antibodies binding to SKBR3) compared to PBMC co-cultured with SKBR3 alone. CpG ODN type C augmented in vitro anti-SKBR3 Ab synthesis. These studies show that Her2Bi-armed ATCco-cultured with PBMC enhanced nonspecific cytotoxicity and induced in vitro specific antibody synthesis directed at SKBR3 cells. Evidence that Her2Bi armed ATC can induce a vaccination response is supported by dendritic cell (DC) loading experiments in which aATC were transiently mixed with SKBR3 cells to generate tumor lysate. IL-4 and GM-CSF generated DC were exposed to the lysate for 24 h, washed, and co-cultured with fresh PBMC for 14 and 21 days. At the end of co-culture, cytotoxicity assays against SKBR3 increased markedly whereas cytotoxicity directed at Daudi targets was low. In addition, there were considerable levels of Abs directed to SKBR3 in the supernatants of PBMC co-cultured with DC loaded with SKBR3 lysate, but not with DC loaded with SKBR3-culture media or RPMI alone. These studies established that the lysate produced as a result of aATC cytotoxicity against SKBR3 is immunogenic for DC. In summary, when PBMC were co-cultured with DC exposed to SKBR3 lysate, there was induction of specific cytotoxicity and in vitro tumor specific Abs synthesis. Together with experiments involving primary co-cultures of irradiated aATC, PBMC, and SKBR3, our studies show that there are both non-specific and specific cellular and humoral responses generated as a result of co-culture with Her2Bi armed ATC. These studies provide evidence that aATC infusions can induce both specific and non-specific cellular, humoral, and cytokine responses from the endogenous immune systems of patients. Please credit the grants R01CA92344, 5P30CA022453-25, 1819 from Michigan Economic Development Corporation and 6066-06 from Leukemia and Lymphoma Society
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Alari-Pahissa, Elisenda, Michelle Ataya, Ilias Moraitis, Miriam Campos-Ruiz, Mireia Altadill, Aura Muntasell, Anna Moles, and Miguel López-Botet. "NK cells eliminate Epstein-Barr virus bound to B cells through a specific antibody-mediated uptake." PLOS Pathogens 17, no. 8 (August 20, 2021): e1009868. http://dx.doi.org/10.1371/journal.ppat.1009868.
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Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.
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Noël, Alexandra, David G. Ashbrook, Fuyi Xu, Stephania A. Cormier, Lu Lu, James P. O’Callaghan, Shyam K. Menon, Wenyuan Zhao, Arthur L. Penn, and Byron C. Jones. "Genomic Basis for Individual Differences in Susceptibility to the Neurotoxic Effects of Diesel Exhaust." International Journal of Molecular Sciences 23, no. 20 (October 18, 2022): 12461. http://dx.doi.org/10.3390/ijms232012461.
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Air pollution is a known environmental health hazard. A major source of air pollution includes diesel exhaust (DE). Initially, research on DE focused on respiratory morbidities; however, more recently, exposures to DE have been associated with neurological developmental disorders and neurodegeneration. In this study, we investigated the effects of sub-chronic inhalation exposure to DE on neuroinflammatory markers in two inbred mouse strains and both sexes, including whole transcriptome examination of the medial prefrontal cortex. We exposed aged male and female C57BL/6J (B6) and DBA/2J (D2) mice to DE, which was cooled and diluted with HEPA-filtered compressed air for 2 h per day, 5 days a week, for 4 weeks. Control animals were exposed to HEPA-filtered air on the same schedule as DE-exposed animals. The prefrontal cortex was harvested and analyzed for proinflammatory cytokine gene expression (Il1β, Il6, Tnfα) and transcriptome-wide response by RNA-seq. We observed differential cytokine gene expression between strains and sexes in the DE-exposed vs. control-exposed groups for Il1β, Tnfα, and Il6. For RNA-seq, we identified 150 differentially expressed genes between air and DE treatment related to natural killer cell-mediated cytotoxicity per Kyoto Encyclopedia of Genes and Genomes pathways. Overall, our data show differential strain-related effects of DE on neuroinflammation and neurotoxicity and demonstrate that B6 are more susceptible than D2 to gene expression changes due to DE exposures than D2. These results are important because B6 mice are often used as the default mouse model for DE studies and strain-related effects of DE neurotoxicity warrant expanded studies.
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Troilo, Arianna, Alessia Grassi, and Mario Milco D'Elios. "Intrinsic factor Recognition Promotes Th17/Th1 Autoimmune Gastric Inflammation in Patients with Pernicious Anemia." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 129.12. http://dx.doi.org/10.4049/jimmunol.202.supp.129.12.
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Abstract The intrinsic factor (IF) is the major humoral autoantigen in pernicious anemia (PA)/autoimmune gastritis. Although many studies have examined the autoantibody response to intrinsic factor and H+,K+-ATPase, no information is available on possible pathogenic mechanisms mediated by IF-specific gastric T cells. The aim of this study was to investigate IF-specific T cells in the gastric mucosa of PA patients and define their functional properties. In vivo-activated T cells from the infiltrates of the gastric mucosa of PA patients were isolated and cloned. We investigated the ability of the gastric T-cell clones to proliferate and produce cytokines in response to IF, as well as their helper function for antibody production and their cytolytic potential. For the first time we provide evidence that gastric mucosa of PA patients harbour a high proportion (20%) of autoreactive activated CD4+ T-cell clones that specifically recognize IF. Most of these clones (94%) showed a Th17 or Th1 profile. Virtually all IF-specific clones produced TNFα, IL-21 and provided substantial help for B-cell immunoglobulin production. Most mucosa-derived IF-specific T-cell clones expressed perforin-mediated cytotoxicity and induced Fas-Fas ligand-mediated apoptosis in target cells. Altogether, our results suggest that chronic autoantigen-induced T cell– dependent B-cell activation is responsible for the local synthesis of IF autoantibodies found in the sera and indicate that activation of IF-specific Th17 and Th1 T cells in the gastric mucosa represent a key effector mechanism in PA suggesting that the Th17/Th1 pathway may represent a novel target for the prevention and treatment of the disease.
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Eyvani, Haniyeh, and Xinna Zhang. "Abstract 1371: Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1371. http://dx.doi.org/10.1158/1538-7445.am2022-1371.
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Abstract Immune response is a critical anti-tumor response to overcome tumorigenesis. In line with this, immune checkpoint blockade shows tremendous anti-tumor efficacy by enhancing cytotoxicity of CD8+ cytotoxic T lymphocytes (CTLs). However, cancer cells develop different mechanisms of immune evasion, among which suppression of immune recognition of somatic mutations via tumor antigen is prevalent. In fact, 65% to 90% of the cancers show reduced MHC-I antigen presentation. There is, nonetheless, little achievement toward the therapeutic approaches to restore antigen presentation on tumor cells. Colorectal cancer (CRC), the second deadliest cancer in U.S., shows increasing incidence and mortality rates over the past years. Standard conventional CRC treatments, surgery, chemotherapy and radiotherapy, often lead to many side effects due to their non-specificity and cytotoxicity. While immunotherapy is very promising, only a small population of CRC patients respond to the current immune checkpoint inhibitors. In this study, our objective was to harness a novel immunological approach to target CRC tumors. We developed a bioinformatics tool to create a pool of genes whose expression negatively correlates with cytotoxicity of CD8+ CTLs in the tumor microenvironment of CRC patients. Given the central role of antigen presentation in activating CD8+ CTLs, we further screened these genes based on the scope of their effects on antigen presentation. To this end, we developed stable clones of OVA expressing MC38 cells (MC38OVA) where OVA-derived peptide, SIINFEKL, will be bound to MHC-I complex for cell surface presentation. 4 genes with highest capacity to alter MHC-I cell surface levels were identified. Our consequent in vivo experiments and in vitro cytotoxicity assays identified that “Rho-GEF” gene was the top actionable target with striking suppressive effects on tumor growth. TCGA database analysis showed Rho-GEF is upregulated in 22% of CRC patients accompanied by a poor prognosis. Rho-GEF knockdown (KD) significantly inhibited tumor growth in immunocompetent mice whereas it only showed minor effects on immunocompromised mice, suggesting that the Rho-GEF may modulate tumor growth by regulating the interaction of tumor cells with non-tumor cells such as immune cells. Consistent with the major contribution of antigen presentation in promoting CD8+ CTLs activity, TNFα, IFNγ, and GZMB levels in CD8+ T cells isolated from Rho-GEF KD MC38-derived tumors showed a significant increase compared to the wild type tumors. Taken together, we introduced Rho-GEF as a potential clinical target involved in CRC immune evasion by modulating antigen presentation and CD8+ CTLs anti-tumor responses. Citation Format: Haniyeh Eyvani, Xinna Zhang. Rho-GEF hampers CD8+T cells-mediated immunosurveillance by reducing antigen presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1371.
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Lortz, S., S. Lenzen, and I. Mehmeti. "Impact of scavenging hydrogen peroxide in the endoplasmic reticulum for β cell function." Journal of Molecular Endocrinology 55, no. 1 (June 24, 2015): 21–29. http://dx.doi.org/10.1530/jme-15-0132.
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Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H2O2). Because of the high rate of insulin biosynthesis and the low expression of H2O2-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H2O2concentration might be very high. As the role of this H2O2in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H2O2removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H2O2significantly with a threefold increase of the EC50value for H2O2. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H2O2reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H2O2toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H2O2in the ER in spite of a reduced insulin transcription and processing.
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Wen, Lu, Gregory T. Wolf, Monil Shah, David B. Page, Lynn Sadowski-Mason, Mark Prince, Jeffrey Moyer, Alfred E. Chang, and Qiao Li. "IRX-2 therapy with PD-L1 blockade in immunocompetent animal model." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e14149-e14149. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e14149.
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e14149 Background: Multiple therapeutic components in IRX-2 including IL-2, IL-1, IFN-γ, TNFα, IL-6, GM-CSF, and IL-8 work synergistically to activate various immune cells including T cells, dendritic cells, and natural killer cells to recognize and kill tumors. Previous Phase 1 and Phase 2a clinical trials in head and neck cancer with IRX-2 have shown a benign safety profile. Immunologically stimulating host immunity using IRX-2, while simultaneously blocking PD-1/PD-L1-mediated immune suppression, may have the potential to enhance the outcome of current immunotherapy using IRX-2 alone. Methods: Healthy C3H mice were inoculated with SCC7 murine head and neck cancer cells. Fourteen days after inoculation, the tumor-bearing mice were treated with subcutaneous injection of IRX-2 once a week for 3 weeks, followed with anti-mPD-L1(i.p.) injection after each IRX-2 administration. Tumor size and survival were monitored for the therapeutic efficacy evaluation. Two weeks after the last IRX-2 administration, all mice were sacrificed and spleen, blood and residual tumor samples were harvested for immune function assays. Results: High levels of PD-L1 were expressed on SCC7 cell line and fresh tumors. The FCM data suggested that PD-L1 was preferentially expressed on the ALDHhigh SCC7 cancer stem cells. IRX-2 alone significantly inhibited SCC7 tumor growth, and the anti-tumor efficacy of IRX-2 was significantly enhanced by anti-mPD-L1 injection in a dose dependent manner in the immunocompetent hosts. Serum IgG from animals subjected to IRX-2 plus anti-mPD-L1 treatment mediated significantly (p < 0.05) higher cytotoxicity on SCC7 cells via ADCC than the IgG from animals treated with IRX-2 alone. CD8+ lymphocytes isolated from purified, activated and expanded CD3+ splenic T cells harvested from the animals subjected to IRX-2+anti-mPD-L1 treatment mediated significantly (p < 0.05) higher cytotoxicity than those subjected to IRX-2 or anti-PD-L1 mono-therapy against SCC7 tumor cells. Conclusions: Co-administration of IRX-2 and anti-PD-L1 promoted more powerful antitumor immunity with both B cell and T cell modulation. The combination treatment increased the specific IgG production that could kill tumor cells via ADCC, and enhanced the T cell CTL activity. Therefore, IRX-2 therapy plus anti-PD-L1 administration may represent a promising novel strategy to treat cancer patients.
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Gurunathan, Sangiliyandi, Muniyandi Jeyaraj, Hyeonwoo La, Hyunjin Yoo, Youngsok Choi, Jeong Tae Do, Chankyu Park, Jin-Hoi Kim, and Kwonho Hong. "Anisotropic Platinum Nanoparticle-Induced Cytotoxicity, Apoptosis, Inflammatory Response, and Transcriptomic and Molecular Pathways in Human Acute Monocytic Leukemia Cells." International Journal of Molecular Sciences 21, no. 2 (January 9, 2020): 440. http://dx.doi.org/10.3390/ijms21020440.
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The thermoplasmonic properties of platinum nanoparticles (PtNPs) render them desirable for use in diagnosis, detection, therapy, and surgery. However, their toxicological effects and impact at the molecular level remain obscure. Nanotoxicology is mainly focused on the interactions of nanostructures with biological systems, particularly with an emphasis on elucidating the relationship between the physical and chemical properties such as size and shape. Therefore, we hypothesized whether these unique anisotropic nanoparticles could induce cytotoxicity similar to that of spherical nanoparticles and the mechanism involved. Thus, we synthesized unique and distinct anisotropic PtNPs using lycopene as a biological template and investigated their biological activities in model human acute monocytic leukemia (THP-1) macrophages. Exposure to PtNPs for 24 h dose-dependently decreased cell viability and proliferation. Levels of the cytotoxic markers lactate dehydrogenase and intracellular protease significantly and dose-dependently increased with PtNP concentration. Furthermore, cells incubated with PtNPs dose-dependently produced oxidative stress markers including reactive oxygen species (ROS), malondialdehyde, nitric oxide, and carbonylated protein. An imbalance in pro-oxidants and antioxidants was confirmed by significant decreases in reduced glutathione, thioredoxin, superoxide dismutase, and catalase levels against oxidative stress. The cell death mechanism was confirmed by mitochondrial dysfunction and decreased ATP levels, mitochondrial copy numbers, and PGC-1α expression. To further substantiate the mechanism of cell death mediated by endoplasmic reticulum stress (ERS), we determined the expression of the inositol-requiring enzyme (IRE1), (PKR-like ER kinase) PERK, activating transcription factor 6 (ATF6), and activating transcription factor 4 ATF4, the apoptotic markers p53, Bax, and caspase 3, and the anti-apoptotic marker Bcl-2. PtNPs could activate ERS and apoptosis mediated by mitochondria. A proinflammatory response to PtNPs was confirmed by significant upregulation of interleukin-1-beta (IL-1β), interferon γ (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL-6). Transcriptomic and molecular pathway analyses of THP-1 cells incubated with the half maximal inhibitory concentration (IC50) of PtNPs revealed the altered expression of genes involved in protein misfolding, mitochondrial function, protein synthesis, inflammatory responses, and transcription regulation. We applied transcriptomic analyses to investigate anisotropic PtNP-induced toxicity for further mechanistic studies. Isotropic nanoparticles are specifically used to inhibit non-specific cellular uptake, leading to enhanced in vivo bio-distribution and increased targeting capabilities due to the higher radius of curvature. These characteristics of anisotropic nanoparticles could enable the technology as an attractive platform for nanomedicine in biomedical applications.
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Dahl, Haley C., Mohammed Kanchwala, Shayna E. Thomas-Jardin, Amrit Sandhu, Preethi Kanumuri, Afshan F. Nawas, Chao Xing, Chenchu Lin, Daniel E. Frigo, and Nikki A. Delk. "Chronic IL-1 exposure drives LNCaP cells to evolve androgen and AR independence." PLOS ONE 15, no. 12 (December 16, 2020): e0242970. http://dx.doi.org/10.1371/journal.pone.0242970.
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Chronic inflammation promotes prostate cancer (PCa) initiation and progression. We previously reported that acute intereluekin-1 (IL-1) exposure represses androgen receptor (AR) accumulation and activity, providing a possible mechanism for IL-1-mediated development of androgen- and AR-independent PCa. Given that acute inflammation is quickly resolved, and chronic inflammation is, instead, co-opted by cancer cells to promote tumorigenicity, we set out to determine if chronic IL-1 exposure leads to similar repression of AR and AR activity observed for acute IL-1 exposure and to determine if chronic IL-1 exposure selects for androgen- and AR-independent PCa cells. We generated isogenic sublines from LNCaP cells chronically exposed to IL-1α or IL-1β. Cells were treated with IL-1α, IL-1β, TNFα or HS-5 bone marrow stromal cells conditioned medium to assess cell viability in the presence of cytotoxic inflammatory cytokines. Cell viability was also assessed following serum starvation, AR siRNA silencing and enzalutamide treatment. Finally, RNA sequencing was performed for the IL-1 sublines. MTT, RT-qPCR and western blot analysis show that the sublines evolved resistance to inflammation-induced cytotoxicity and intracellular signaling and evolved reduced sensitivity to siRNA-mediated loss of AR, serum deprivation and enzalutamide. Differential gene expression reveals that canonical AR signaling is aberrant in the IL-1 sublines, where the cells show constitutive PSA repression and basally high KLK2 and NKX3.1 mRNA levels and bioinformatics analysis predicts that pro-survival and pro-tumorigenic pathways are activated in the sublines. Our data provide evidence that chronic IL-1 exposure promotes PCa cell androgen and AR independence and, thus, supports CRPCa development.
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Nakamura, Mano, Elmira Amiri Souri, Gabriel Osborn, Roman Laddach, Jitesh Chauhan, Chara Stavraka, Sara Lombardi, et al. "IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype." Cancers 12, no. 11 (November 15, 2020): 3376. http://dx.doi.org/10.3390/cancers12113376.
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IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNFα, MCP-1, IL-10, CXCL-10, IL-1β, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.
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Cha, Mi Young, Hyunkyung Yu, Bu-Nam Jeon, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Joo-Yeon Chung, et al. "Abstract 1868: Novel immune checkpoint inhibitor: In vivo and in vitro efficacy of anti-CNTN4 antibody, GENA-104A16 in the models with increased CNTN4 expression." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1868. http://dx.doi.org/10.1158/1538-7445.am2023-1868.
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Abstract We previously suggested that contactin 4 (CNTN4) negatively regulates T cell activity through binding with amyloid precursor protein on T cells. In addition, we confirmed that CNTN4 is highly expressed in the various types of tumors such as gallbladder, pancreas, stomach, endometrium, liver, prostate, and bladder cancer and melanoma with positive rates over 70% through immunohistochemistry analysis. However, murine cancer cells expressed CNTN4 at a very low level, differently from human tumors or cancer cells. Therefore, it was limited in assessing the anti-tumor efficacy by targeting CNTN4 in syngeneic mice models. Following this background, we have prepared CT26 murine cancer cells overexpressing CNTN4 (CT26/CNTN4) and evaluated anti-tumor efficacy by treating GENA-104A16, the anti-CNTN4 humanized monoclonal antibody in the CT26/CNTN4 syngeneic mice model. The treatment of GENA-104A16 induced fairly improved in vivo efficacy in the model compared to the CT26 WT model. Moreover, to identify the in vivo immune modulatory function of GENA-104A16, we analyzed the proportion of tumor-infiltrated immune cells in CT26/CNTN4 tumors through flow cytometry and single-cell analysis. As a result, the population of Treg cells decreased in the tumors of GENA-104A16-treated mice. In addition, the total amount of tumor-infiltrated immune cells increased by 2-fold, CD4+ T cells by 4-fold, and cytotoxic CD8+ T cells by 1.5-fold in tumors of mice administrated with GENA-104A16. We also performed transcriptomic analysis on samples collected from IgG-treated CT26 WT tumors and CT26/CNTN4 tumors treated with IgG or GENA-104A16. Comparative analysis of gene expression using the Gene Set Enrichment Analysis Hallmark showed that the gene sets of TNFα signaling, IFNγ response, inflammatory response, and apoptosis were decreased in CT26/CNTN4 tumors than in CT26 WT tumors. And the majority of the two gene sets, TNFα signaling and IFNγ response in CT26/CNTN4 tumors, increased by the administration of GENA-104A16. These results indicate that the overexpression of CNTN4 reduces the activity of tumor-infiltrating lymphocytes, resulting in decreased cytokine secretion, which is restored by administering GENA-104A16. Furthermore, we investigated in vitro T cell-mediated cytotoxicity of GENA-104A16 on U2OS (human osteosarcoma cell) endogenously expressing CNTN4 together with CNTN4-knocked out U2OS. GENA-104A16 increased T cell-mediated cytotoxicity when CNTN4-expressing U2OS cancer cells were co-cultured with T cells. Collectively, the function of CNTN4 in negative immune regulation was confirmed repeatedly through the present studies. And the expression level of CNTN4 on tumors was essential for the anti-tumor activity of GENA-104A16. Therefore, the expression of CNTN4 could be a potential biomarker for monitoring clinical responses to anti-CNTN4 therapy. Citation Format: Mi Young Cha, Hyunkyung Yu, Bu-Nam Jeon, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Joo-Yeon Chung, Gihyeon Kim, Changho Park, Kyung Mi Park, Hansoo Park. Novel immune checkpoint inhibitor: In vivo and in vitro efficacy of anti-CNTN4 antibody, GENA-104A16 in the models with increased CNTN4 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1868.
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Coquant, G., D. Aguanno, A. Peyrottes, L. Brot, S. Thenet, J. P. Grill, L. De Sordi, and P. Seksik. "P053 3-Oxo-C12:2, a quorum sensing molecule from the human gut, exerts anti-inflammatory effects through a bitter taste receptor." Journal of Crohn's and Colitis 14, Supplement_1 (January 2020): S160—S161. http://dx.doi.org/10.1093/ecco-jcc/jjz203.182.
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Abstract Background Acyl-homoserine lactones (AHLs) are quorum sensing molecules involved in bacterial communication network and can also have an impact on host’s cells. We recently showed the presence of AHLs in the gut ecosystem and identified one that has never been described: 3-oxo-C12:2. This molecule was decreased in faecal samples of inflammatory bowel disease (IBD) patients, especially during flare, and its absence was correlated to dysbiosis. 3-Oxo-C12:2 is structurally close to an AHL well described and synthesised by P. aeruginosa, 3-oxo-C12. We intent to describe 3-oxo-C12:2 effects on gut inflammation and to identify which signalling pathways are involved. Given its analogous structure to 3-oxo-C12, we hypothesised that 3-oxo-C12:2 can interact with the same cellular partners, in particular a bitter taste receptor, called T2R138, which is a GPCR expressed by immune and epithelial gut cells. Methods To test our hypothesis, we used murine macrophages cell line RAW264.7, stimulated by interferon-γ (IFN-γ; 20 U/ml) and lipopolysaccharide (LPS; 10 ng/ml). Inflammatory response was monitored by measuring cytokine secretion (TFNα, IL-6 and IL-10) via ELISA. Protein expression was assessed by Western blot. Probenecid, a known allosteric inhibitor for T2R138, was used to study T2R138 role in AHL signalling. Cytotoxicity was checked by measuring LDH release. Results In steady state, challenged by 3-oxo-C12:2, RAW264.7 macrophages showed no change in cytokine expression (TNFα, IL-6 and IL-10). After LPS/IFN-γ activation, we observed a decrease in the ratio of secreted TNFα/IL-10 when cells are exposed to 3-oxo-C12:2, in a dose-dependent manner: 15 μM (−30%, p < 0.05), 25 μM (−50%, p < 0.001) and 50 μM (−65%, p < 0.0001). This reflects an anti-inflammatory effect, without increasing cytotoxicity. Those immune-modulatory effects were lost in presence of Probenecid. Moreover, the amount of T2R138 protein was not changed in the presence of Probenecid and/or 3-oxo-C12:2. Therefore, the loss of anti-inflammatory effects with the inhibitor was not due to a decrease of the receptor expression in our experimental conditions. Conclusion 3-Oxo-C12:2 exerts a dose-dependent anti-inflammatory effect on murine immune cells. This response is partly mediated by the bitter taste receptor T2R138. This receptor is a potential target of our AHL. We are currently studying via a multiplex assay the cytokine response after exposing cells to 3-oxo-C12:2. Studying the signalling between the receptor and the anti-inflammatory response would allow us to understand better the inter-kingdom dialogue between microbiota and the host and to what extend AHLs are involved. We are grateful for the FRM for the financial support (ECO201806006843)
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Dolstra, Harry, Henriette Levenga, Ingrid Overes, Johanna C. M. Vos, Agnes van Horssen-Zoetbrood, Robbert van der Voort, Pieter de Mulder, and T. M. de Witte. "Aberrant Expression in Human Epithelial Cancers of the P2X5-Encoded Minor Histocompatibility Antigen LRH-1: Implications for Graft-Versus-Tumor Immunity Against Solid Tumors." Blood 110, no. 11 (November 16, 2007): 1795. http://dx.doi.org/10.1182/blood.v110.11.1795.1795.
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Abstract Allogeneic stem cell transplantation (SCT) in combination with donor lymphocyte infusion (DLI) is an experimental treatment for patients with metastatic solid tumors. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived T cells eliminate malignant cells via recognition of minor histocompatibility antigens (MiHA). To reduce accompanying GVHD, it is crucial to identify MiHA which are selectively expressed on hematopoietic cells and solid tumor cells. Previously, we identified a hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest.2005:115:3506–3516). Here, we report that LRH-1, in addition to its hematopoietic cell-restricted expression, is aberrantly expressed on epithelial tumor cell lines. We observed that P2X5 mRNA is significantly expressed in 14 out of 42 (33%) solid tumor cell lines tested by real-time quantitative RT-PCR. We detected P2X5 transcripts in 3 out of 11 renal cell carcinoma cell lines, 2 out of 4 melanoma cell lines, 3 out of 7 colorectal carcinoma cell lines, 4 out of 10 brain tumor cell lines and 2 out of 10 breast cancer cell lines. To determine whether P2X5 mRNA expression in solid tumor cell lines results in susceptibility to lysis by LRH-1-specific CTL, we performed flow cytometry-based cytotoxicity assays using P2X5-expressing tumor cell lines. Based on LRH-1 genotyping analysis, we selected six solid tumor cell lines for the cytotoxicity studies. Remarkably, LRH-1-specific CTL efficiently lysed and inhibited the growth of DAOY brain tumor cells up to 3 days of co-culture. The renal cell carcinoma cell lines SKRC-33 and SKRC-18 and the melanoma cell line BLM were also susceptible to LRH-1 CTL-mediated lysis, although less effectively. However, pre-incubation of these tumor cell lines with IFNγ and TNFα significantly increased the susceptibility to LRH-1-specific CTL and resulted in complete target cell lysis. Furthermore, these cytokine-stimulated cell lines induced higher levels of CTL degranulation as determined by CD107a staining. No cytotoxicity was observed against LRH-1-negative FM3 melanoma and SKRC-24 renal cell carcinoma cell lines. These findings illustrate that the GVT reactivity observed in solid tumors after allogeneic SCT may be selectively enhanced by LRH-1-specific immunotherapy.
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Stephens, Deborah M., Kyle A. Beckwith, Carolyn Cheney, Xiaokui Mo, Joseph M. Flynn, Jeffrey A. Jones, Kami J. Maddocks, Karl-Heinz Heider, Natarajan Muthusamy, and John C. Byrd. "BI 836826, a Novel Fc-Engineered Antibody in Combination with Phosphoinositide-3-Kinase Inhibitor for Treatment of High Risk Chronic Lymphocytic Leukemia." Blood 124, no. 21 (December 6, 2014): 4681. http://dx.doi.org/10.1182/blood.v124.21.4681.4681.
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Abstract Novel chronic lymphocytic leukemia (CLL) therapies target phosphoinositide 3-kinase (PI3K; idelalisib) and Bruton's tyrosine kinase (BTK; ibrutinib). Despite their success, certain high-risk groups, notably those with dysfunctional P53, demonstrate lower response rates than other CLL groups. As such, combination therapies could potentially overcome this deficiency. As CD37-mediated signaling resulted in direct cytotoxicity that was enhanced with PI3K inhibition (Lapalombella 2012), we aimed to combine BI 836826, a novel IgG1 chimerized and Fc-engineered anti-CD37 mAb, with BCR-pathway inhibitors to possibly enhance clinical efficacy in high-risk CLL patients and overcome potential inhibition of ADCC. Consistent with previous report, BI 836826 displays both ADCC and direct pro-apoptotic activity (Heider 2011). We assessed the potential effect of idelalisib and ibrutinib on CD37 expression and NK-cell function. Following 24hr incubation of primary CLL cells (n=4) with idelalisib, ibrutinib, or DMSO, there was no significant difference in surface expression of CD37 (fold change ~ 1.01; p > 0.16 for all comparisons). To ensure that NK-cell Fc-receptor function was unimpaired by BCR-pathway inhibitors, we conducted CD107ab degranulation assays (n=5). Data revealed that Fc-receptor engagement of BI 836826 and NK-cell lytic degranulation was not abrogated by idelalisib or ibrutinib (mean CD107ab expression: DMSO = 10.14%; idelalisib = 5.67%; ibrutinib = 2.41%). In combination with idelalisib, BI 836826 was still capable of eliciting an effective NK-cell response superior to rituximab (difference = 4.51%; p < 0.0001). To further qualify NK-cell function, TNFα (n = 6) or INFγ (n = 12) release by NK cells after pretreatment with idelalisib or ibrutinib was evaluated. Similarly, cytokine release was not completely inhibited by idelalisib and ibrutinib; and BI 836826 was still capable of inducing cytokine release. To confirm the potential added benefit of combination therapy, we conducted 51Cr release ADCC assays with primary CLL cells and healthy allogeneic NK-cells (Fig1) or CLL patient autologous NK-cells (Fig2). Results confirmed that neither ibrutinib nor idelalisib could ablate BI 836826-induced NK-cell ADCC. However, there was a more pronounced inhibition of NK-cell ADCC with ibrutinib. As previously demonstrated (Kohrt 2014), NK-cell ADCC mediated by rituximab was almost completely abrogated by ibrutinib, likely secondary to off-target interleukin-2-inducible T-cell kinase inhibition (Dubovsky 2013). Our preliminary data show that signaling induced by BI 836826 may demonstrate the same enhanced direct cytotoxicity with PI3K inhibition as previously reported with other CD37 mediated signaling agents (Lapalombella 2012). Evaluation of direct pro-apoptotic activity of BI 836826 (0.1ug/mL) in combination with idelalisib (1uM) on direct cell death (24hr incubation) demonstrated enhanced activity in the combination setting with similar activity in primary patient CLL cells with dysfunctional P53 (n=6; Fig3A) and functional P53 (n=4; Fig3B). In conclusion, these results demonstrate that idelalisib could potentially be used in combination with BI 836826, which has the added benefit of directly killing inhibitor-resistant P53-null primary CLL cells and lacks complete inhibitor-induced ADCC ablation. Figure 1. Allogeneic ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 1. Allogeneic ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 2. Autologous ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 2. Autologous ADCC after Pretreatment of NK-cells with BTK inhibitor (Ibrutinib) or PI3K inhibitor (Idelalisib) Figure 3. BI 836826 Direct Cytotoxicity in High- and Low-Risk CLL Patients Figure 3. BI 836826 Direct Cytotoxicity in High- and Low-Risk CLL Patients Disclosures Off Label Use: Off-label use of idelalisib and BI 836826 in combination for CLL patients. Jones:Pharmacyclics: Consultancy, Research Funding. Heider:Boehringer Ingelheim: Employment.
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Pawlowsky, Lea, Bettina Brauchle, Maryam Kazerani Pasikhani, Ana Ogrinc Wagner, Veit Bücklein, Felix S. Lichtenegger, Roman Kischel, et al. "Impact of p53 Knock-Down on T-Cell Proliferation and T-Cell Mediated Cytotoxicity Against AML Cell Lines Mediated By a CD33 Specific BiTE® Antibody Construct." Blood 134, Supplement_1 (November 13, 2019): 1265. http://dx.doi.org/10.1182/blood-2019-122415.
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Bispecific T-cell engaging (BiTE®) antibody constructs recruit T cells independent of the T-cell receptor specificity. Proof of concept was shown by Blinatumomab, a CD19xCD3 BiTE® antibody construct, for treatment of r/r B-cell precursor ALL. Response rates of 43% led to its approval in 2015, however, the mechanisms of innate and adaptive resistance in non-responders are still incompletely understood. To improve response rates, it is imperative to understand intrinsic and acquired resistance mechanisms. We assessed the relevance of p53 knockdown (kd) in AML cells on cytotoxicity mediated by AMG 330, a CD33 specific BiTE®antibody construct. We initially hypothesized that p53 kd cells would show differential sensitivity to T-cell mediated cytotoxicity. 12 AML cell lines with different p53 status (wt: MV4-11, Molm-13; mut/del: HL60, MONO-MAC 6, K562, Kasumi-1, Kasumi-3, THP-1, SKM-1, NB-4, KG1a) or p53 wt and paired p53 kd cell lines (MV4-11, Molm-13 and OCI-AML3) were cocultured with healthy donor (HD) T cells at an effector: target (E:T) ratio of 1:6 with AMG 330 (5 ng/ml) or a control BiTE®for 3 days. Cytotoxicity was calculated as %specific lysis = 100 × (1 - live CD33+cellsAMG 330/live CD33+cellscBiTE®). To assess T cell proliferation, T cells were stained with the proliferation dye Far Red. For analysis of the costimulatory/coinhibitory expression profile of the paired cell lines, cells were stained for CD80, CD86, CD40L, OX40L, PD-L1, Tim-3 and corresponding isotypes either in steady state or after 72h stimulation with TNFα and IFNγ. To dissect the influence of receptor-ligand interactions versus secretome, we performed transwell experiments. HD T cells and murine Ba/F3 cells (transduced with CD33 and CD86) were cocultured at an E:T ratio of 1:10 in presence of 5 ng/ml AMG 330 or control BiTE®in the lower compartment. In the upper compartment AML cells were cultured, allowing the exchange of soluble factors but no cell-cell contact. Ba/F3 cells were used as a surrogate model to activate T cells and minimize the influence of human checkpoint molecules. To further delineate the influence of p53 kd on T-cell function, we performed transcriptome analysis of the paired AML cell lines. First, 12 AML cell lines were analyzed for AMG 330-mediated cytotoxicity upon coculture with T cells. No difference based on p53 status was observed (p53 wt vs p53 mut (65.86% vs 53.92%; n=27). However, phenotypic and genetic heterogeneity of AML cell lines could mask the influence of p53 mutation on the pharmacodynamic activity of AMG 330. To exclude these cofounding covariables, we performed cytotoxicity assays using p53 wt and paired p53 kd cell lines. AMG 330-mediated cytotoxicity was significantly higher for p53 wt cell lines compared to p53 kd cell lines (wt vs kd: MV4-11: 89.8% vs 72.3%, p<0.03; Molm-13: 90.0% vs 85.2%, p<0.03; OCI-AML3: 89.5% vs 81.2%, p<0.03). Surprisingly, we observed significantly reduced T-cell proliferation in co-cultures with p53 kd cell lines vs p53 wt cell lines (wt vs kd: MV4-11: 74.0% vs 61.6%, p<0.03; Molm-13: 65.7% vs 56.4%, p<0.03; OCI-AML3: 34.9% vs 26.4%, p<0.03). We further demonstrated that the difference is not due to different expression levels of the target antigen CD33 or the costimulatory/coinhibitory molecules CD80, CD86, CD40L, OX40L, PD-L1 and Tim-3 (n= 4). In transwell experiments, T cells proliferated at a lower rate in the presence of p53 kd AML cells in the upper comportment compared to those in the presence of p53 wt AML cells (MV4-11 p53 wt vs p53 kd: 67.9% vs 50.9%, p<0.03) leading to the conclusion that the inhibition of T-cell proliferation is mediated by soluble factors secreted by AML cells. Bulk RNA sequencing of p53 kd AML cell lines compared to p53 wt revealed downregulation of a co-stimulatory ligand 4-1BBL with log2 fold change of - 0.33, -0.30, -0.39 in Molm-13, MV4-11 and OCI-AML3, respectively. In summary, we have observed lower activity of AMG 330 against p53 kd cell lines compared to p53 wt AML cell lines. This correlated with a decrease in T-cell proliferation indicating that the genetic profile of the AML cells modulates T-cell function. We hypothesize that p53 kd AML cells suppress T-cell proliferation, thereby reducing the E:T ratio and redirected cytotoxicity. In a next step, we will try to identify possible secreted mediators of T-cell modulation by mass. We will also characterize primary patient samples for their T-cell-inhibiting capacities, e.g. p53-mutated disease. Disclosures Lichtenegger: Roche: Employment. Kischel:AMGEN Research (Munich) GmbH: Employment, Equity Ownership. Metzeler:Celgene: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Otsuka: Honoraria. Andreeff:NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy. Daver:Celgene: Consultancy; Otsuka: Consultancy; Jazz: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Immunogen: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Agios: Consultancy; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Servier: Research Funding; Incyte: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Glycomimetics: Research Funding; Hanmi Pharm Co., Ltd.: Research Funding; Astellas: Consultancy; NOHLA: Research Funding. Subklewe:Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Morphosys: Research Funding; Janssen: Consultancy; Oxford Biotherapeutics: Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Research Funding; Miltenyi: Research Funding.
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Hideshima, Teru, Constantine S. Mitsiades, Hiroshi Ikeda, Dharminder Chauhan, Noopur Raje, Gullu Gorgun, Mariateresa Fulciniti, et al. "Bcl6 as a Novel Therapeutic Target in Multiple Myeloma (MM)." Blood 114, no. 22 (November 20, 2009): 295. http://dx.doi.org/10.1182/blood.v114.22.295.295.
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Abstract Abstract 295 BCL6 (B- cell lymphoma 6) is a proto-oncogene encoding a transcriptional repressor and regulates germinal center B cell differentiation. It is deregulated by chromosomal translocations or aberrant somatic hypermutation in a subset of diffuse large B cell lymphomas (Polo, JM. et al. PNAS, 2007). Importantly, small peptide inhibitor of BCL6 mediates cytotoxicity in primary human DLBCL cells both in vitro and in vivo, without effecting normal lymphoid tissue (Cerchietti, LC. et al. Blood, 2009). In this study, we examined modulation of Bcl6 expression and its sequelae in human MM cells in the context of bone marrow (BM) microenvironment. By Western blot analysis, constitutive expression of Bcl6 was either undetectable or very weakly expressed in all MM cell lines (MM.1S, MM.1R, RPMI8226, RPMI-LR5 H929, OPM1 and OPM2) except U266 cells. Importantly, however, Bcl6 expression was markedly upregulated by co-culture of MM cells with bone marrow stromal cells (BMSCs). Since U266 has high baseline phopho-STAT3 and BMSC-induced Bcl6 upregulation in other MM cell lines was associated with phosphorylation of STAT3, we hypothesized that JAK/STAT3 signaling might mediate Bcl6 expression in MM cells in the context of BM microenvironment. Indeed, anti-IL-6 neutralizing Ab significantly blocked BMSC co-culture-induced Bcl6 expression. To assess the clinical relevance of Bcl6 expression observed in MM cell lines, we examined its expression in patient MM cells by tissue microarray immunohistochemical analysis, confirming that Bcl6 was strongly positive within the nucleus in all cases. To obtain direct evidence that IL-6 triggered Bcl6 expression, we cultured MM cells with recombinant human IL-6; as expected, IL-6 strongly triggered Bcl6 expression in MM cell lines in a dose- and time-dependent fashion whereas other cytokines (ie, IGF-1, VEGF, IL-3) did not. Importantly, Bcl6 was also induced by IL-6 in primary tumor cells from MM patients. Real time RT-PCR confirmed that both BMSC co-culture and IL-6 treatment upregulated expression of Bcl6 mRNA. Furthermore, oncostatin-M, which also induces phospho-STAT3, similarly upregulated Bcl6. Conversely, pan-JAK inhibitor AG490 and STAT3 siRNA markedly downregulated Bcl6 expression in U266 cells, associated with downregulation of phospho-STAT3. Taken together, these results indicate that gp130 family member cytokines upregulate Bcl6 expression in MM cells via JAK/STAT3 signaling. We also found that TNFα also triggered Bcl6 in MM cell lines (MM.1S, RPMI8226, OPM1 and OPM2) and primary MM tumor cells; however, TNFα-induced upregulation of Bcl6 was independent on STAT3 activation. Importantly, IKKβ inhibitor MLN120B significantly reduced Bcl6 expression in MM cells triggered by TNFα. Taken together, these results suggested that TNFα-induced Bcl6 expression is mediated via the canonical NF-κB signaling pathway. Finally, to examine the biologic significance of Bcl6 inhibition in MM cells, we downregulated Bcl6 expression using lentiviral shRNA, in the presence or absence of IL-6. Importantly, downregulation of Bcl6 significantly decreased the number of MM cells, associated with decreased S and G2/M phase on cell cycle analysis. Our results therefore suggest that Bcl-6 expression is modulated via both JAk/STAT3 and NF-κB pathways in MM cells. Therefore Bcl6 represents a novel therapeutic target in MM.since targeting these cascades could downregulate Bcl-6 expression, and inhibit growth of MM cells in the bone marrow milieu. Disclosures: Raje: Millennium: Speakers Bureau. Munshi:: . Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium: Research Funding.
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Martinez-Planes, Elena, Miguel G. Fontela, Cecilia Ramello, and Daniel Abate-Daga. "Abstract 1770: CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK)." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1770. http://dx.doi.org/10.1158/1538-7445.am2023-1770.
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Abstract CD28-derived co-stimulation modules are frequently used as part of the signaling domain of chimeric antigen receptors (CAR). These modules contain well characterized tyrosine-containing moieties (YMNM and PYAP) that become phosphorylated upon CAR activation, and result in IL-2 secretion and T cell expansion. Previous work by our group has shown that an additional tyrosine residue, located in the C-terminus of CD28 (Y218), was also phosphorylated upon CAR activation. The kinase responsible for this phosphorylation remains unknown, but in silico studies point towards the IL-2-inducible T-cell kinase (ITK), the protein-tyrosine kinase TEC and the bone marrow tyrosine kinase (BMX). In this work we sought to elucidate the molecular mechanism and functional relevance of CD28-Y218 phosphorylation in CAR-T cells.To analyze the kinetics of Y218 phosphorylation, prostate stem cell antigen (PSCA)-specific second-generation CAR-T cells were stimulated through co-culture with HPAC pancreatic cancer cells for 0, 1, 10, 30 and 60 minutes. Phosphorylation of CD28 Y218, Zap70, PLCγ and Vav1 was analyzed by Western blotting. To determine the role of ITK in CD28-218 phosphorylation: 1) PSCA-specific CAR-T cells were treated with ITK inhibitors 10 μM (BMS-509744 or Ibrutinib) for 24h and 2) ITK-deficient Jurkat cells expressing a PSCA-specific CAR were cocultured with HPAC cells for 0 or 10 min. To determine the functional relevance of this phosphorylation event, we generated mutant CARs where the tyrosine residue was replaced with a non-phosphorylatable amino acid (Y218F) and evaluated its effects in vivo and in vitro. CAR-T cell cytotoxicity was monitored using the xCELLigence Real Time Cytotoxicity Assay (RTCA) and cytokine production was assessed following overnight co-culture with target cells using the ELLA system (Biotechne, CA, USA). To evaluate the in vivo antitumor efficacy of PSCA-specific CAR-T cells, NSG mice were injected (s.c.) with 1 HPAC cells and infused 14 days later with 5 CAR-T cells (i.v.). Tumor growth was monitored for 35 days.Our results show that phosphorylation of CD28-Y218 occurs after the phosphorylation of early TCR signaling mediators, reaching maximum levels around 10-30 min post-stimulation. Pharmacological inhibition and genomic ablation of ITK impairs CD28-Y218 phosphorylation, suggesting an essential role of ITK in this signaling event. We observed that mutation of the Y218 residue did not affect CAR expression, in vitro cytotoxicity, or INFγ production. However, this mutation was associated with reduced IL-2 and TNFα secretion. In vivo, this mutation completely abrogated the therapeutic effect of CAR-T cells in a pancreatic cancer model. In conclusion, these results indicate that ITK is required for CD28-Y218 phosphorylation, a signaling event that plays an important role in CAR-T cell function, with direct impact on IL-2 secretion and antitumor efficacy. Citation Format: Elena Martinez-Planes, Miguel G. Fontela, Cecilia Ramello, Daniel Abate-Daga. CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1770.
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Freeman, Ciara Louise, Franck Morschhauser, Laurie H. Sehn, Mark Dixon, Richard Houghton, Thierry Lamy, Gunter Fingerle-Rowson, et al. "Pattern of Cytokine Release in Patients with Chronic Lymphocytic Leukemia Treated with Obinutuzumab and Possible Relationship with Development of Infusion Related Reactions (IRR)." Blood 124, no. 21 (December 6, 2014): 4674. http://dx.doi.org/10.1182/blood.v124.21.4674.4674.
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Abstract Background: Infusion related reactions (IRR) are commonly seen with administration of biologic therapies but remain relatively poorly understood. Rituximab is thought to induce cell death upon binding to CD20 primarily by complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Obinutuzumab (GA101, Gazyva®, Gazyvaro®) is a new generation humanised IgG1 Type II anti-CD20 mAb with contrasting properties to rituximab. The Fc portion has been glycoengineered (afucosylated) to increase binding affinity to Fcgamma receptor 3(FcγRIII). These features result in higher direct cell killing activity, enhanced ADCC and phagocytosis (ADP) and less complement activation compared with rituximab. Early experience with rituximab has shown that release of pro-inflammatory cytokines occurs with initial administration and is higher in patients experiencing IRRs compared to those who are not. We report on the cytokine release in a subset of 38 patients with chronic lymphocytic leukemia (CLL) treated with obinutuzumab monotherapy pooled from the earlier Phase I (GAUSS) trial (NCT00576758) and Phase I/II (GAUGUIN) trial (NCT00517530). Methods: Eligible patients for both studies had relapsed/refractory CLL with no alternative, higher priority therapy available. All patients were treated sequentially with intravenous (IV) obinutuzumab monotherapy and were universally pre-medicated with an oral anti-pyretic (paracetamol/acetaminophen) and an IV anti-histamine 30 minutes prior to starting the infusion. Steroid pre-medication was not mandated in either study. Cytokine serum samples (quantitatively measured using the Becton-Dickinson (BD) Cytometric Bead Array) were taken from each patient prior to each obinutuzumab infusion, mid-way through the infusion, at the end of infusion and 2-5 hours after completion. Samples for complement analysis were also taken at these same time points (C3/C4 analysed using Siemens BNII Nephelometer, C3a/C5a using BD Pharmingen ELISA kit). Peripheral blood samples for immunophenotyping were also taken at baseline, prior to and at the end of each infusion. The development of an IRR with the first infusion was defined as the occurrence of related signs and symptoms within 24 hours of administration of antibody and graded according to the CTCAE guidelines v4.0. Results: Of the 38 patients treated, 35 developed symptoms of IRR (Grade 1/2=25, Grade 3/4=10) with the first infusion accompanied by a rapid decrease in circulating CD19+ B cells, a drop in the measurable natural killer (NK) CD16+56+ cells and an increase of pro-inflammatory cytokines IL6, IL8, TNFα and IFNγ (see figures 1 and 2). There were no meaningful differences between pre-treatment baseline cytokine levels across all subgroups. At the mid-infusion time-point, patients with absolute lymphocyte count (ALC) at baseline ≥50x109/L released higher levels of IL6 (mean IL6(log10) 3.16 vs 2.41), IL-8 (mean IL8(log10)3.57 vs 2.91), TNFα (mean TNFα(log10) 2.59 vs 1.92), IFNγ (mean IFNγ(log10)2.38 vs 1.8) and IL10 (mean IL10(log10) 2.03 vs 1.69) than those with ALC <50x109. Patients who had higher grade IRR (≥grade 3) had higher baseline values for lymphocyte count and β-2microglobulin, lower baseline platelet count and higher Binet stage than those without severe grade IRR, however none of these differences were statistically significant at p≤0.05. . Markers of complement activation, C5a and C3a, did not increase and C3/C4 levels remained stable across all groups. Cytokine release was limited to the first infusion of obinutuzumab only and did not recur with subsequent infusions (Fig . 1). Conclusions: This sub-analysis demonstrates that the first obinutuzumab administration triggers immediate and strong release of cytokines (IL6, IL8, TNFα, IFNγ and IL10) in association with rapid destruction of circulating B cells in patients with CLL. The close temporal relationship between release of pro-inflammatory cytokines (especially IL6 and IL8) and development of IRRs, in addition to the lack of complement activation induced by obinutuzumab, suggest that cytokine release is part of the IRR pathophysiology. Intervention strategies targeting cytokines may therefore be a promising strategy to reduce the incidence and severity of IRRs. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Freeman: Roche Pharmaceuticals: clinical research fellowship supported by Roche Pharmaceuticals (secondment from Bart's) Other. Off Label Use: Presentation may refer to mitigation strategies such as the use of tocilizumab in patients with cytokine release syndrome. Morschhauser:Bayer: Honoraria; Mundipharma: Honoraria; Genentech: Honoraria; Spectrum: Honoraria; Gilead: Honoraria. Sehn:Roche: Research Funding. Dixon:Roche Pharmaceuticals: Employment. Houghton:Roche Pharmaceuticals: Employment. Fingerle-Rowson:Roche Pharmaceuticals: Employment. Wassner-Fritsch:Roche Pharmaceuticals: Employment. Hallek:Roche Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau. Salles:Roche Pharmaceuticals: Honoraria, Research Funding. Cartron:Roche: Consultancy, Honoraria.
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Fiedler, Walter, Cristiana Sessa, Luca Gianni, Sara Cresta, Henning Schulze-Bergkamen, Jens Weidmann, Sara De Dosso, et al. "First-in-human phase I study of CetuGEX, a novel anti-EGFR monoclonal antibody (mAb) with optimized glycosylation and antibody dependent cellular cytotoxicity." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 3008. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.3008.
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3008 Background: Epidermal growth factor receptor (EGFR) is a validated target in cancer. EGFR antagonists in clinical use do not exploit the full potential of this target. CetuGEX is an IgG1 mAb against EGFR. Fully human and optimized glycosylation lead to a 10- to 250-fold improvement of ADCC-mediated tumor cell killing in all FcγRIIIa allotypes and lack of immunogenic carbohydrate-chains, compared to cetuximab. Methods: Eligible patients with advanced solid tumors, progressing after standard treatment, were enrolled into this phase I, first-in-human, multicenter, single agent dose escalation trial. PK, PD and immunological parameters were assessed. Endpoints were safety and tolerability and secondarily pharmacokinetics, immunogenicity and anti-tumor activity. Results: 41 patients were treated on a q1w (8 dose levels from 12 to 1,370 mg flat dose), or q2w (990 mg flat) schedule. 25 pts had received at least 8 weekly doses (per protocol population [PP]).The most frequently observed drug-related AE were nausea (20%), vomiting (20%), hypertension (20%), almost all low grade and acneiform dermatitis (25%), only grade 1 or 2. Infusion-related reactions (IRR), virtually restricted to the first infusion, were associated with cytokine secretion: IL-6, IL-8, TNFα, IFNγ and IP-10 as marker of macrophage activation. Optimization of infusion scheme and premedication reduced IRRs in severity and frequency from 76% to 57% mainly of low grade. Blood NK cells were reduced as sign of redistribution. Activity was seen over all dose levels. One patient with NSCLC achieved a complete response. One patient with metastatic colorectal cancer had a partial response, another 2 patients with esophageal and gastric cancer without measurable disease at study entry had marked improvement of symptoms and normalization of tumor markers. Additional 15 pts had stable disease lasting from 8 weeks to over a year, including several minor responses, leading to a clinical benefit rate of 46% (19/41) in the overall and 76% (19/25) in the PP population. PK supports q1w and q2w dosing. Conclusions: CetuGEX shows clear signs of activity and acceptable toxicity. Phase II will soon be initiated. Clinical trial information: NCT01222637.
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Hu, Guangan, Quanju Zhao, Lai Shi, Nan Bing, and Dong Zhang. "Abstract 3433: Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3433. http://dx.doi.org/10.1158/1538-7445.am2022-3433.
Full textAbstract:
Abstract Background: Genome-wide association studies have identified HLA-G and LILRB1/2 loci associated with cancer risk. LILRB1 and LILRB2 are distinct inhibitory immune checkpoint receptors that recognize MHC-I ligands, particularly immunosuppressive HLA-G. LILRB1-mediated inhibition can lead to impairment of T-cell cytotoxicity and preventing the efficient engulfment of tumor cells by macrophages as a “don’t eat me” signal. LILRB2 is expressed primarily by myeloid cells, especially in the tumor microenvironment, as an important immunosuppressive signal to inhibit the stimulation of an immune response. A dual antagonist antibody targeting both LILRB1 and LILRB2 could be more effective for cancer immunotherapy than targeting either LILRB1 or LILRB2 individually. Methods: Mice were immunized with recombinant D1-D2 domain of LILRB1 and LILRB2 proteins. Resulted heavy chain and light chain variable regions of IgG were PCR amplified from splenocytes and bone marrow plasma cells. PCR amplicons were built into yeast libraries. Yeast displayed scFv were screened for LILRB1 and LILRB2 binding. The dual binders were converted to effector silent human IgG1 and evaluated for ability to block HLA-G binding to LILRB1/RB2. The dual binder and dual blocker were further assessed in series of assays using human T cells and various primary myeloid cell subsets, including human monocyte-derived macrophages (hMDMs), tumor-associated macrophages (TAMs), dendritic cells (DCs) and monocytes. Results: We have discovered a panel of anti-LILRB1/2 antibodies that bind to both LILRB1 and LILRB2 with high affinity and block the interactions to HLA-G efficiently. The anti-LILRB1/2 antibodies inhibit both LILRB1 and LILRB2 signaling and modulate the function of immune cells. Anti-LILRB1/LILRB2-mediatted LILRB1 blockade: 1) enhances phagocytosis of tumor cells by macrophages; 2) releases HLA-G mediated inhibition on TCR activation in Jurkat T cells over-expressing LILRB1; 3) promotes the activation of human primary T cells stimulated with allogenic DCs. Anti-LILRB1/LILRB2-mediatted LILRB2 blockad:1) enhances TNFα expression and inhibits IL10 expression in LPS-stimulated human PBMC; 2) reprograms both hMDMs and TAMs induced by tumor cells to polarize toward a more inflammatory phenotype, including enhanced T cell responses to stimulation by DCs and macrophages, increased expression of M1 markers and secreted proinflammatory cytokine TNFα, decreased expression of M2 markers and secreted anti-inflammatory cytokine IL10. Conclusions: We have generated dual antagonist anti-LILRB1/LILRB2 antibodies. The anti-LILRB1/LILRB2 can reprogram suppressive myeloid cells to a stimulatory state, enhance phagocytotic function of myeloid cells, and promote the activation of lymphoid cells. The data support further development of anti-LILRB1/LILRB2 as an immunotherapeutic for solid tumor malignancies. Citation Format: Guangan Hu, Quanju Zhao, Lai Shi, Nan Bing, Dong Zhang. Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3433.
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Teng, Xinyi, Hao Zhang, Zuyu Liang, Mi Shao, Xiujian Wang, Lijuan Ding, Jiazhen Cui, Yongxian Hu, and He Huang. "Galectin-9 from Bone Marrow Mesenchymal Stromal Cells Mediates Immunosuppression on Chimeric Antigen Receptor T Cells." Blood 134, Supplement_1 (November 13, 2019): 5609. http://dx.doi.org/10.1182/blood-2019-131680.
Full textAbstract:
Objective: Chimeric antigen receptor T cells (CART) emerged as a robust therapeutic approach for refractory or relapsed B acute lymphoblastic leukaemia (B-ALL) in recent years. However, 30-50% of patients experienced leukaemia relapse within 1 year after CART cells therapy. Bone marrow derived mesenchymal stromal cells (MSCs) have been demonstrated to have immunosuppressive properties on T cell-mediated immune responses, but the impacts of MSCs on CART cells are not clear. Here we address the role of galectin-9 secreted from MSCs in immunosuppression of CART cells. Methods: MSCs and T cells were isolated from bone marrow and peripheral blood of healthy donors, respectively, and CD19 targeted CART cells containing 4-1BB costimulatory construction were prepared as previously reported. The proliferation of CART cells was evaluated by cell counting and Ki-67 expression. The expression of PD1, TIM3, LAG3 and FasL was detected by flow cytometry. The cytotoxicity of CART cells was determined by luciferase-based assays. Transwell was used to assess the contribution of soluble factors. The mRNA expression of COX-2, IL-6, IL-10, TGFβ and galectin-9 was detected by real time PCR. We used the lentivirus-based shRNA interference to assess the role of Galetin-9 on immunosuppression of CART cells. Results: In this work, we discovered that CAR-T cells co-cultured with MSCs for 72 hours in vitro showed a decreased proliferation rate and Ki-67 expression. And the inhibition effect became more significant with the increase in the proportion of MSCs (Fig.a,b,c,d). Notably, the expression of inhibitory receptor TIM3 upregulated remarkably while no change of PD1, LAG3 and FasL were observed(Fig.e,f). We routinely use luciferase-based cytotoxic assay for functional testing. By this measure, we found that with the increase in the proportion of MSC, CAR-T cells behave even weaker cytotoxic effect, achieving lower cytolysis rate of Nalm6 cells(Fig.g).To further determine the possible factors that contribute to the changes mentioned above, CART cells were separated from MSCs by transwell. As expected, the phenomenon of inhibited CART cells proliferation and cytotoxicity, increased expression of TIM3 was observed as well, although without direct contact of CART cells with MSCs. The contribution of soluble factors was in consideration and further experiments of real time PCR revealed that COX-2 and galectin-9 were strongly induced by CART cells and pro-inflammatory cytokines(IFNγ, TNFα). In light of that galectin-9 is the ligand of inhibitory receptor TIM3, which increased obviously in the presence of MSCs, we speculated that galectin-9 might be responsible for immunosuppression of CART cells. A knockdown approach with shRNA demonstrated the immunosuppressive activity of galectin-9 deficient MSCs on CART cells decreased significantly. We provided experimental evidence that galectin-9 may contribute to MSC mediated immunosuppression on CART cells by binding to its receptor TIM-3. Conclusion: Our findings demonstrated for the first time that galectin-9 is involved in MSC mediated immunosuppression on CART cells, and represented a potential therapeutic target for enhancing the efficacy of CART cells and reducing the incidence of leukaemia relapse after CART cells therapy. Key words: Chimeric antigen receptor T cells; mesenchymal stromal cells; immunosuppression; galectin-9. Fig. a. Co-culture of CART cells and MSCs. b,c,d. Cell proliferation and Ki-67 expression of CART cells co-cultured with MSCs at different ratios. e,f. Expression of TIM3, PD1, LAG3, FasL of CART cells co-cultured with MSCs at different ratios. g. Cytotoxicity of CART cells co-cultured with MSCs at different ratios (CART without MSC, CART:MSC=1:5, CART:MSC=1:10, CART:MSC=1:20). Figure Disclosures No relevant conflicts of interest to declare.