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P3.07-004 GSDMD Is Required for Effector CD8+ T Cell Responses to Lung Cancer Cell
November 2017 response of immune system against cancer with control for the cancer cells-derived exosomes. Because dendritic cells (DCs) play a key role in immune reactions to activate T cells against cancer cells by cancer antigen presentation at cellular membrane, DCs have been used in clinical trials as cellular mediators for therapeutic vaccination. It has reported that the exosomes released from vaccinated DCs are responsible for the persistence of antigen presentation. Cancer cells derived exosomes play an immunosuppressive. We considered that whether DCs-derived exosomes could induce suppress cancer cells and more effective response of immune system against cancer with control for the cancer cells-derived exosomes. Method: DCs were generated from bone marrow cells in C57BL/6J by stimulation with GM-CSF and IL-4 mice for 6 days. Murine lung cancer cell line (3LL) was cultured in RPMI1640 medium containing 10%FCS. 3LL cells-derived exosomes and DCs-derived ones were isolated by ultracentrifugation methods and exosomes purification kit (Qiagen). 3LL cells were injected to C57BL/6J mice by intraperitoneal administration. DCs, DCs-exosomes or 3LL-exosmes were weekly administrated to cancer bearing mice. Tumor growth inhibition by exosomes was evaluated measurement of luciferase activity by in vivo image analyzing system. Result: DCs and DCs-derived exosomes inhibited lung cancer cell growth, on the other hand, lung cancer derived-exosomes increased in compared with DCs, DCs-exosomes and non-treated. Conclusion: For cancer immunotherapy, DC-exosomes and cancer-exosomes play important roles in cancer immune reactions. Further examination, we are going on analyze immunosuppressive molecules possessing cancer cell-derived exosomes, and immune activation molecules in DCs-exosomes. Keywords: dendritic cells, Immunotherapy, Exosome
P3.07-004 GSDMD Is Required for Effector CD8+ T Cell Responses to Lung Cancer Cell T. Lv,1 G. Xi,1 H. Liu,1 F. Zhang,1 Y. Song2 1Jinling Hospital, Nanjing/ CN, 2Respiratory Medicine, Jinling Hospital, Nanjing/CN Background: Cytotoxic T lymphocytes (CTLs) play a critical role in protection against intracellular pathogens and tumor. To induce target cell death, CTL mainly use two major contact-dependent cytotoxic pathways that are dependent on Fas ligand (FasL) and lytic granules. CTLs eliminate malignantly transformed cells principally by releasing the contents of cytotoxic granules into the immune synapse formed with their target cell. The granule serine proteases, known as granzymes (Gzms), induce apoptosis after they are delivered into the target cell cytoplasm by the pore-forming granule protein perforin. Therefore, we hypothesized that other pore-forming protein, especially those can form pores from within mammalian cells, may be implicated in the target cell killing process of CTL. Since GSDMD is a recently discovered pore-forming protein whose N-terminal domain can insert the inner leaflet of the cell membrane and form extensive pores, we speculate that GSDMD may participate in the CTL attack. GSDMD is a recently discovered pyroptosis executioner in monocyte, whose N-terminal domain can insert the inner leaflet of the cell membrane and form extensive pores. Although the role of GSDMD in the pyroptosis has been clear, the function of GSDMD in other biological system remains elusive. In the present study, we investigated the role of GSDMD during CTL responses to NSCLC cancer cells. Method: 3LL and H1299 cells were cultured in RPMI-1640 (HyClone, USA) supplemented with 10% fetal bovine serum, Ovalbumin-expressing 3LL cells (3LL-OVA) were generated by transfection with a lentiviral plasmids harboring cytosolic chicken ovalbumin. C57BL/6 mice and TCR-transgenic OT-1 mice. Mouse CD8+ T cell isolation and stimulation, Human CD8+ T cell isolation and stimulation, Real-time PCR analysis, Western blot analysis, Immunofluorescence cell staining, Immunohistochemistry, Lentiviral vectors transduction, In vitro cytotoxicity assays, Bioinformatics analysis, Result: We showed that GSDMD expression was consistently correlated with CD8+ T cell markers in TCGA cohorts. The expression of
Abstracts
S2299
GSDMD protein could be detected in the tumor infiltrating lymphocyte. GSDMD cleavage increased both in the OT-1 CTLs and the human activated CD8+ T cells. Moreover, Colocalization of GSDMD with granzymeB was observed in proximity of immune synapse. GSDMD deficiency reduced the cytolytic capacity of human CD8+ T cells. Conclusion: These results identified a previously unknown role of GSDMD in CTL and demonstrated that GSDMD is required for an optimal CTL response to lung cancer cell. Keywords: Immune response, NSCLC, Gasdermin family, GSDMD, CTL
P3.07-005 Activation of Toll-like Receptor-2 Promotes Proliferation in Human Lung Adenocarcinoma Cells P. Kohtz, S. Kalatardi, A. Sjoberg, X. Meng, D. Fullerton, M. Weyant Surgery, University of Colorado - Denver, Aurora, CO/US Background: Toll-like receptors (TLR) have been implicated in tumor progression by affecting the immune response in the tumor microenvironment. The role of these receptors in the growth and proliferation of human lung cancer cells is not known. We analyzed human lung cancer cells for the presence of TLR-2, their proliferation response to a TLR-2 agonist and the role of NFkB in TLR-2 activation. Method: Human non-small cell lung cancer (NSCLC) cell lines A549 and 1650 (adenocarcinoma), H1299 metastatic NSCLC and H125 adenosquamous were cultured using standard techniques. Standard Western blotting techniques determined baseline TLR-2 protein levels. Cells were treated with Pam3CSK4, a specific agonist of TLR-2, at doses of 0ug/ml, 5ug/ml, and 10ug/ml for 72 hours, and then the MTS assay was used for proliferation assessment. All cell lines were treated with 10 ug/ml Pam3CSK4 for 48 hours and Western blot analysis determined the level of increase in NFkB phosphorylation. Cells were treated with NFkB inhibitor, Bay11-7082, at doses of 1 uM, 5 uM and 25 uM with concomitant treatment of 10 ug/ml Pam3CSK4 over 5 hours. NFkB inhibition was evaluated by Western blot analysis. Result: Protein analysis demonstrated consistently detectable levels of TLR-2 in each cell line (Fig 1a). Proliferation was significantly promoted after Pam3CSK4 treatment in human lung adenocarcinoma cell lines (p¼.03), but not in the other cell lines (p<0.05) (Fig 1b). NFkB phosphorylation was increased with TLR2-agonist treatment in adenocarcinoma, decreased in non-adenocarcinoma cells and decreased with specific inhibition of NFkB subunit p65 (Fig1c). Conclusion: TLR-2 is consistently present and detectable on human lung adenocarcinoma cells. TLR-2 activation results in increased proliferation in human lung adenocarcinoma cells and this effect appears to be specific to adenocarcinoma cells. These effects are dependent on the NFkB signaling pathway. These findings may suggest TLR-2 to be a possible therapeutic target in human lung cancer. Keywords: lung adenocarcinoma, Tumor immunology, Toll-like receptor
P3.07-004 GSDMD Is Required for Effector CD8+ T Cell Responses to Lung Cancer Cell T. Lv,1 G. Xi,1 H. Liu,1 F. Zhang,1 Y. Song2 1Jinling Hospital, Nanjing/ CN, 2Respiratory Medicine, Jinling Hospital, Nanjing/CN Background: Cytotoxic T lymphocytes (CTLs) play a critical role in protection against intracellular pathogens and tumor. To induce target cell death, CTL mainly use two major contact-dependent cytotoxic pathways that are dependent on Fas ligand (FasL) and lytic granules. CTLs eliminate malignantly transformed cells principally by releasing the contents of cytotoxic granules into the immune synapse formed with their target cell. The granule serine proteases, known as granzymes (Gzms), induce apoptosis after they are delivered into the target cell cytoplasm by the pore-forming granule protein perforin. Therefore, we hypothesized that other pore-forming protein, especially those can form pores from within mammalian cells, may be implicated in the target cell killing process of CTL. Since GSDMD is a recently discovered pore-forming protein whose N-terminal domain can insert the inner leaflet of the cell membrane and form extensive pores, we speculate that GSDMD may participate in the CTL attack. GSDMD is a recently discovered pyroptosis executioner in monocyte, whose N-terminal domain can insert the inner leaflet of the cell membrane and form extensive pores. Although the role of GSDMD in the pyroptosis has been clear, the function of GSDMD in other biological system remains elusive. In the present study, we investigated the role of GSDMD during CTL responses to NSCLC cancer cells. Method: 3LL and H1299 cells were cultured in RPMI-1640 (HyClone, USA) supplemented with 10% fetal bovine serum, Ovalbumin-expressing 3LL cells (3LL-OVA) were generated by transfection with a lentiviral plasmids harboring cytosolic chicken ovalbumin. C57BL/6 mice and TCR-transgenic OT-1 mice. Mouse CD8+ T cell isolation and stimulation, Human CD8+ T cell isolation and stimulation, Real-time PCR analysis, Western blot analysis, Immunofluorescence cell staining, Immunohistochemistry, Lentiviral vectors transduction, In vitro cytotoxicity assays, Bioinformatics analysis, Result: We showed that GSDMD expression was consistently correlated with CD8+ T cell markers in TCGA cohorts. The expression of
Abstracts
S2299
GSDMD protein could be detected in the tumor infiltrating lymphocyte. GSDMD cleavage increased both in the OT-1 CTLs and the human activated CD8+ T cells. Moreover, Colocalization of GSDMD with granzymeB was observed in proximity of immune synapse. GSDMD deficiency reduced the cytolytic capacity of human CD8+ T cells. Conclusion: These results identified a previously unknown role of GSDMD in CTL and demonstrated that GSDMD is required for an optimal CTL response to lung cancer cell. Keywords: Immune response, NSCLC, Gasdermin family, GSDMD, CTL
P3.07-005 Activation of Toll-like Receptor-2 Promotes Proliferation in Human Lung Adenocarcinoma Cells P. Kohtz, S. Kalatardi, A. Sjoberg, X. Meng, D. Fullerton, M. Weyant Surgery, University of Colorado - Denver, Aurora, CO/US Background: Toll-like receptors (TLR) have been implicated in tumor progression by affecting the immune response in the tumor microenvironment. The role of these receptors in the growth and proliferation of human lung cancer cells is not known. We analyzed human lung cancer cells for the presence of TLR-2, their proliferation response to a TLR-2 agonist and the role of NFkB in TLR-2 activation. Method: Human non-small cell lung cancer (NSCLC) cell lines A549 and 1650 (adenocarcinoma), H1299 metastatic NSCLC and H125 adenosquamous were cultured using standard techniques. Standard Western blotting techniques determined baseline TLR-2 protein levels. Cells were treated with Pam3CSK4, a specific agonist of TLR-2, at doses of 0ug/ml, 5ug/ml, and 10ug/ml for 72 hours, and then the MTS assay was used for proliferation assessment. All cell lines were treated with 10 ug/ml Pam3CSK4 for 48 hours and Western blot analysis determined the level of increase in NFkB phosphorylation. Cells were treated with NFkB inhibitor, Bay11-7082, at doses of 1 uM, 5 uM and 25 uM with concomitant treatment of 10 ug/ml Pam3CSK4 over 5 hours. NFkB inhibition was evaluated by Western blot analysis. Result: Protein analysis demonstrated consistently detectable levels of TLR-2 in each cell line (Fig 1a). Proliferation was significantly promoted after Pam3CSK4 treatment in human lung adenocarcinoma cell lines (p¼.03), but not in the other cell lines (p<0.05) (Fig 1b). NFkB phosphorylation was increased with TLR2-agonist treatment in adenocarcinoma, decreased in non-adenocarcinoma cells and decreased with specific inhibition of NFkB subunit p65 (Fig1c). Conclusion: TLR-2 is consistently present and detectable on human lung adenocarcinoma cells. TLR-2 activation results in increased proliferation in human lung adenocarcinoma cells and this effect appears to be specific to adenocarcinoma cells. These effects are dependent on the NFkB signaling pathway. These findings may suggest TLR-2 to be a possible therapeutic target in human lung cancer. Keywords: lung adenocarcinoma, Tumor immunology, Toll-like receptor