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The Transcription Factor NFATc2 Controls Apoptosis and Activation of T Cells by IL-6 in Colitis
Abstracts were observed in proximal and distal jejunum as well as ileum of NOD vs. BALB/c mice. Furthermore, a significantly lower ratio of intraepithelial to lamina propria CD3+ cells was found in NOD mice. Interestingly, NOD mice fed both diabetes-protective diets displayed lower number of CD3+ cells within their lamina propria. Our results showed altered villus architecture, immatureactivated enzyme profile of the small intestine, and an impairment of gut mucosal immune system of pre-diabetic NOD compared to BALB/c mice as well as changes in lamina propria T cells of NOD mice fed diabetespreventive diets. doi:10.1016/j.clim.2010.03.197
T.86. Variations of Vaccinia Virus' Replication in Melanoma and Lung Cell Lines Based on Different Expression of IFI6, and IFTMN2 and β-microglobin
Maria Ascierto 1, Andrea Worschech 2, Zoltan Pos 1, Fabio Rossano 4, Paolo Ascierto 3, Ena Wang 1, Francesco Marincola 1. 1National Institutes of Health, Bethesda, MD; 2 Genelox Corporation, San Diego, CA; 3National Cancer Institute Fondazione G. Pascale, Napoli, Italy; 4University Federico II, Napoli, Italy Introduction: Vaccinia virus (VACV) and other lytic viruses are used in oncolytic therapy to selectively colonize tumors causing a tumor regression. The ability of the virus to colonize and replicate in cancer cells mediating tumor regression is strongly dependent by the microenvironment, as well as host's immune system, which is believed to limit the effects of oncolytic therapy by limiting viral replication. Method: To characterize the relationship between baseline immune activation of cancer cells and viral replication, we screened several melanoma (13) and lung cancer (9) cell lines among NCI-60 panel for their permissivity to infection by vaccinia virus (VACV) GLV-1h68, carrying GFP gene. Subsequent microarray analyses were performed for the screening of genes involved in VACV replication. Results: At 18 hours after infection, virus's infectivity and replication were evaluated in all tested cells analyzing GFP expression by FACS analyses. The results were than confirmed monitoring the expression of three virus genes by using TaqMan® Gene Expression Assays. Based on the results obtained, the cell lines were classified in high and low infectivity indices. Transcriptional profiling showed that replication of VACV is strongly limited in melanoma cells constitutively expressing Interferon-alpha protein 6 (IFI6) and in lung cancer expressing high level of interferon induced trasmembrane protein 2 (IFTMN2) and B-microglobulin. Understanding the relationship between basic activation of intracellular innate immunity and VACV replication success of oncolytic therapy in preclinical models may shed important information about the role of immunity in oncolytic therapy and, more broadly, the mechanism(s) leading to immune-mediated, tissuespecific rejection. doi:10.1016/j.clim.2010.03.198
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T.96. Mechanistic Studies of Tolerance in sublingual immunotherapy (SLIT) patients with Dermatophagoides farinae and Timothy grass allergy Neha Reshamwala, Ravi Swamy, Sean Berquist, Tammy Nguyen, Elizabeth Hoyte, Soujanya Vissamsetti, Abirami Sivagnanasundaram, Vivian Saper, Peter Hwang, Richard Moss, Kari Nadeau. Stanford University, Stanford, CA Background: We investigated the mechanisms of cellular tolerance in concurrent Dermatophagoides farinae (DF) and Timothy grass (TG) SLIT in patients ages 5–65. T-cell subsets, function assays, and plasma cytokine profiles were analyzed to test if development of tolerance in treated subjects was associated with specific and/or nonspecific allergens, reducing medication use and the severity of allergic rhinoconjunctivitis (AR/AC) symptoms. Methods: A double-blind 2:1 randomized placebo-controlled study in 30 subjects with multiple allergies including at least DF and TG. Regulatory T cells (Treg) were purified from peripheral blood mononuclear cells from each subject. Treg subsets were analyzed for Foxp3 transcription through QT–PCR; epigenetic analysis of the Foxp3 locus occurred via pyrosequencing technology. Treg function was evaluated in autologous samples. Plasma was analyzed using Luminex 35 plex studies and ELISA. Results: Treg functional assays showed an improvement of Treg function by 2–3 months of therapy. Phenotyping of Treg subsets and levels of plasma-derived tolerogenic immune indicators demonstrated an increase in IL-10 (5.3-fold), and TGB beta (9.5-fold), after 3 months of dosing. In contrast, decreases were seen in IL-4, IL-13, and IL-9, Th2-inflammatory cytokines, by at least 4-fold. Conclusion: This is the first double allergen SLIT trial in children to study immune mechanisms of Treg function which show improved tolerance to offending allergens over non offending allergens. The response to immunotherapy with SLIT may induce up regulation of adaptive or natural regulatory T cells. We have correlated these cellular studies with improvement in subject symptom scores for AR/AC. doi:10.1016/j.clim.2010.03.199
T.100. The Transcription Factor NFATc2 Controls Apoptosis and Activation of T Cells by IL-6 in Colitis Benno Weigmann 1, Hans-Anton Lehr 2, Stefan Rose-John 3, Markus Neurath 1. 1University Hospital Erlangen, Erlangen, Germany; 2Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 3Christian Albrechts University, Kiel, Germany The functional role of T cell transcription factors such as nuclear factor of activated T cells (NFAT) in IBD is poorly understood. The aim was to analyze the role of this signal transduction pathway and its pathogenic significance in UC. Cryosections of patients were analysed immunohistochemically. A higher expression of NFATc2 was found in UC and CD colonic tissue compared to control specimen. Transmitted to the Th2-mediated oxazolone-induced colitis model, NFATc2-production is significantly increased in both
S66 diseases, too. NFATc2-deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice. Interestingly, cryosections of inflamed colonic tissue displayed a higher apoptotic rate in NFATc2 deficient mice compared to control mice, which can be observed by TUNEL assays, caspase3, and Annexin V staining, as well as in lamina propria T cells. Contrary, bcl-2 and bcl-xL were downregulated for induction of apoptosis. This observation was associated with a reduced production of IL-6, IFN-gamma, IL-13, and IL-17 by mucosal T lymphocytes, tested by ELISA assays. Administration of IL-6 blocked the protective effects of the NFATc2 deficiency in experimental colitis, suggesting that NFATc through IL-6 signal transduction plays a direct pathogenic role in vivo. Our data define a unique regulatory role of NFATc2 in colitis by controlling mucosal T cell activation in an IL-6-dependent manner. doi:10.1016/j.clim.2010.03.200
T.101. Microarray and CyTOF Characterization of Plasmacytoid Dendritic Cell Subsets Regina Cheung, Yury Goltsev, Sean Bendall, Gary Nolan, Paul J. Utz. Stanford University, Stanford, CA Plasmacytoid dendritic cells (pDCs) are bone marrow derived cells that secrete type I interferon in response to activation of Toll-like-receptors (TLRs) 7 and 9, which detect nucleic acids of viral or endogenous origin. We have demonstrated that pDCs respond heterogeneously to TLR stimulation and have identified distinct pDC subsets from cultures of FLT3L-derived DCs (FL-DCs). FL-DCs were stimulated with CpG-A or polyuridylic acid precincubated with DOTAP, followed by incubation with brefeldin-A. FLDCs were fixed, permeabilized, and stained for CD11b and B220 to subset for pDCs, and for cytokines TNF, IFNalpha, and IL-6, then analyzed by FACS. Several distinct subsets of pDCs were identified: those staining for a single cytokine; those staining for two cytokines (IL-6 and TNF, IFN-alpha and TNF); and a subset staining for all three. Individual subsets were characterized by transcript profiling and CyTOF, in which inductively coupled plasma mass spectrometry (ICP-MS) detects metal tagged antibodies, to identify surface markers that identify these subsets. Antibodies against candidate surface markers revealed by the transcript profiling were bound via maleimide chemistry with polymer DN3 tag containing multiple metal chelates loaded with lanthanide ions. After stimulation, fixation, and permeabilization, the FL-DCs were stained with a cocktail containing over 30 antibodies, incubated with an iridium DNA intercalator, then analyzed by CyTOF. Here, we present characterization of each FL-DC subset, with a long-term goal to determine the role played by individual subsets in SLE and other autoimmune diseases. doi:10.1016/j.clim.2010.03.201
Abstracts
T.102. Altered T Cell Antigen-specific Receptor Signaling in Tumor Infiltrating T Cells in Follicular Lymphomas Faye Hsu, Jonathan Irish, Karen Sachs, Garry Nolan. Stanford University, Stanford, CA T cell antigen-specific receptor (TCR)-mediated cell activation, together with the presence of certain cytokines, is a major factor for determining T cell function and proliferation. Inefficient T cell activation against cancer cells, even when there are abundant tumor-antigen specific cells, is one of the most critical causes of failed antitumor immunity. As accumulated data suggest that T cells are inactivated in tumor, we hypothesize that the tumorinfiltrating T cells will have impaired TCR-mediated signaling mechanism. Our approach resulted in a detailed kinetic map of phosphorylation of upstream (CD3z, LCK, ZAP70, and SLP76) and downstream proteins (ERK1/2 and p38). When we compared the strength and kinetics of TCR signaling in T cell subsets, we found strikingly similar patterns of phosphoprotein activation. The maximum strength of CD4 T helper cells (CD4+ Foxp3−) signaling was greater than that of CD4 Treg (CD4+ Foxp3+ ) and CD8 T cells (CD8+ ). We also demonstrated that TCR-mediated signaling was significantly reduced in tumor-infiltrating T helper cells, but not the tumor-infiltrating CD8 cells, by measuring multiple downstream factors, as compared to normal peripheral blood T cells. These data suggest impaired T cell activation is at least partly due to failed early signaling events of TCR. doi:10.1016/j.clim.2010.03.202
T.103. Characterization of Mouse Nasal-associated Lymphoid Tissue (NALT) in BALB/c and NOD (non-obese diabetic) Mice Petra Fundová 1, Helena Tlaskalova-Hogenová 2, David Funda 2. 1Central Military Hospital, Prague, Czech Republic; 2 Institute of Microbiology, Czech Academy of Science, Prague, Czech Republic Nasal-associated lymphoid tissue (NALT) is a mucosal immune inductive site at the upper respiratory tract. It may represent an equivalent of gut(G)ALT or bronchial(B)ALT in its location and probably plays an important role in induction of immune responses after i.n. administration of antigens. NOD mouse is a well-established model in type 1 diabetes, and intranasal vaccination represents a promising way in the disease prevention. In this study, we first established an isolation protocol for preparation of cell suspensions of mouse NALT cells. Next, using FACS analysis, we analyzed the composition of NALT in SPF (specific pathogen-free) BALB/c mice and started comparisons with NOD mice. The average yield cells per NALT varied from 1.6×105 to 3.0×105. Cell viability and purity was checked by propidium iodide (PI) and CD45 staining. We evaluated basic subsets of B cells (B1 and B2) and T cells (CD4+, CD8+, and TCRγδ+) and expression of markers such as CD69 and CD62L within T (CD3-, CD4-gated) cells. Similarly, the proportion of regulatory CD4+CD25+CD45RBlow, CD4+CD103+ as
T.86. Variations of Vaccinia Virus' Replication in Melanoma and Lung Cell Lines Based on Different Expression of IFI6, and IFTMN2 and β-microglobin
Maria Ascierto 1, Andrea Worschech 2, Zoltan Pos 1, Fabio Rossano 4, Paolo Ascierto 3, Ena Wang 1, Francesco Marincola 1. 1National Institutes of Health, Bethesda, MD; 2 Genelox Corporation, San Diego, CA; 3National Cancer Institute Fondazione G. Pascale, Napoli, Italy; 4University Federico II, Napoli, Italy Introduction: Vaccinia virus (VACV) and other lytic viruses are used in oncolytic therapy to selectively colonize tumors causing a tumor regression. The ability of the virus to colonize and replicate in cancer cells mediating tumor regression is strongly dependent by the microenvironment, as well as host's immune system, which is believed to limit the effects of oncolytic therapy by limiting viral replication. Method: To characterize the relationship between baseline immune activation of cancer cells and viral replication, we screened several melanoma (13) and lung cancer (9) cell lines among NCI-60 panel for their permissivity to infection by vaccinia virus (VACV) GLV-1h68, carrying GFP gene. Subsequent microarray analyses were performed for the screening of genes involved in VACV replication. Results: At 18 hours after infection, virus's infectivity and replication were evaluated in all tested cells analyzing GFP expression by FACS analyses. The results were than confirmed monitoring the expression of three virus genes by using TaqMan® Gene Expression Assays. Based on the results obtained, the cell lines were classified in high and low infectivity indices. Transcriptional profiling showed that replication of VACV is strongly limited in melanoma cells constitutively expressing Interferon-alpha protein 6 (IFI6) and in lung cancer expressing high level of interferon induced trasmembrane protein 2 (IFTMN2) and B-microglobulin. Understanding the relationship between basic activation of intracellular innate immunity and VACV replication success of oncolytic therapy in preclinical models may shed important information about the role of immunity in oncolytic therapy and, more broadly, the mechanism(s) leading to immune-mediated, tissuespecific rejection. doi:10.1016/j.clim.2010.03.198
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T.96. Mechanistic Studies of Tolerance in sublingual immunotherapy (SLIT) patients with Dermatophagoides farinae and Timothy grass allergy Neha Reshamwala, Ravi Swamy, Sean Berquist, Tammy Nguyen, Elizabeth Hoyte, Soujanya Vissamsetti, Abirami Sivagnanasundaram, Vivian Saper, Peter Hwang, Richard Moss, Kari Nadeau. Stanford University, Stanford, CA Background: We investigated the mechanisms of cellular tolerance in concurrent Dermatophagoides farinae (DF) and Timothy grass (TG) SLIT in patients ages 5–65. T-cell subsets, function assays, and plasma cytokine profiles were analyzed to test if development of tolerance in treated subjects was associated with specific and/or nonspecific allergens, reducing medication use and the severity of allergic rhinoconjunctivitis (AR/AC) symptoms. Methods: A double-blind 2:1 randomized placebo-controlled study in 30 subjects with multiple allergies including at least DF and TG. Regulatory T cells (Treg) were purified from peripheral blood mononuclear cells from each subject. Treg subsets were analyzed for Foxp3 transcription through QT–PCR; epigenetic analysis of the Foxp3 locus occurred via pyrosequencing technology. Treg function was evaluated in autologous samples. Plasma was analyzed using Luminex 35 plex studies and ELISA. Results: Treg functional assays showed an improvement of Treg function by 2–3 months of therapy. Phenotyping of Treg subsets and levels of plasma-derived tolerogenic immune indicators demonstrated an increase in IL-10 (5.3-fold), and TGB beta (9.5-fold), after 3 months of dosing. In contrast, decreases were seen in IL-4, IL-13, and IL-9, Th2-inflammatory cytokines, by at least 4-fold. Conclusion: This is the first double allergen SLIT trial in children to study immune mechanisms of Treg function which show improved tolerance to offending allergens over non offending allergens. The response to immunotherapy with SLIT may induce up regulation of adaptive or natural regulatory T cells. We have correlated these cellular studies with improvement in subject symptom scores for AR/AC. doi:10.1016/j.clim.2010.03.199
T.100. The Transcription Factor NFATc2 Controls Apoptosis and Activation of T Cells by IL-6 in Colitis Benno Weigmann 1, Hans-Anton Lehr 2, Stefan Rose-John 3, Markus Neurath 1. 1University Hospital Erlangen, Erlangen, Germany; 2Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 3Christian Albrechts University, Kiel, Germany The functional role of T cell transcription factors such as nuclear factor of activated T cells (NFAT) in IBD is poorly understood. The aim was to analyze the role of this signal transduction pathway and its pathogenic significance in UC. Cryosections of patients were analysed immunohistochemically. A higher expression of NFATc2 was found in UC and CD colonic tissue compared to control specimen. Transmitted to the Th2-mediated oxazolone-induced colitis model, NFATc2-production is significantly increased in both
S66 diseases, too. NFATc2-deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice. Interestingly, cryosections of inflamed colonic tissue displayed a higher apoptotic rate in NFATc2 deficient mice compared to control mice, which can be observed by TUNEL assays, caspase3, and Annexin V staining, as well as in lamina propria T cells. Contrary, bcl-2 and bcl-xL were downregulated for induction of apoptosis. This observation was associated with a reduced production of IL-6, IFN-gamma, IL-13, and IL-17 by mucosal T lymphocytes, tested by ELISA assays. Administration of IL-6 blocked the protective effects of the NFATc2 deficiency in experimental colitis, suggesting that NFATc through IL-6 signal transduction plays a direct pathogenic role in vivo. Our data define a unique regulatory role of NFATc2 in colitis by controlling mucosal T cell activation in an IL-6-dependent manner. doi:10.1016/j.clim.2010.03.200
T.101. Microarray and CyTOF Characterization of Plasmacytoid Dendritic Cell Subsets Regina Cheung, Yury Goltsev, Sean Bendall, Gary Nolan, Paul J. Utz. Stanford University, Stanford, CA Plasmacytoid dendritic cells (pDCs) are bone marrow derived cells that secrete type I interferon in response to activation of Toll-like-receptors (TLRs) 7 and 9, which detect nucleic acids of viral or endogenous origin. We have demonstrated that pDCs respond heterogeneously to TLR stimulation and have identified distinct pDC subsets from cultures of FLT3L-derived DCs (FL-DCs). FL-DCs were stimulated with CpG-A or polyuridylic acid precincubated with DOTAP, followed by incubation with brefeldin-A. FLDCs were fixed, permeabilized, and stained for CD11b and B220 to subset for pDCs, and for cytokines TNF, IFNalpha, and IL-6, then analyzed by FACS. Several distinct subsets of pDCs were identified: those staining for a single cytokine; those staining for two cytokines (IL-6 and TNF, IFN-alpha and TNF); and a subset staining for all three. Individual subsets were characterized by transcript profiling and CyTOF, in which inductively coupled plasma mass spectrometry (ICP-MS) detects metal tagged antibodies, to identify surface markers that identify these subsets. Antibodies against candidate surface markers revealed by the transcript profiling were bound via maleimide chemistry with polymer DN3 tag containing multiple metal chelates loaded with lanthanide ions. After stimulation, fixation, and permeabilization, the FL-DCs were stained with a cocktail containing over 30 antibodies, incubated with an iridium DNA intercalator, then analyzed by CyTOF. Here, we present characterization of each FL-DC subset, with a long-term goal to determine the role played by individual subsets in SLE and other autoimmune diseases. doi:10.1016/j.clim.2010.03.201
Abstracts
T.102. Altered T Cell Antigen-specific Receptor Signaling in Tumor Infiltrating T Cells in Follicular Lymphomas Faye Hsu, Jonathan Irish, Karen Sachs, Garry Nolan. Stanford University, Stanford, CA T cell antigen-specific receptor (TCR)-mediated cell activation, together with the presence of certain cytokines, is a major factor for determining T cell function and proliferation. Inefficient T cell activation against cancer cells, even when there are abundant tumor-antigen specific cells, is one of the most critical causes of failed antitumor immunity. As accumulated data suggest that T cells are inactivated in tumor, we hypothesize that the tumorinfiltrating T cells will have impaired TCR-mediated signaling mechanism. Our approach resulted in a detailed kinetic map of phosphorylation of upstream (CD3z, LCK, ZAP70, and SLP76) and downstream proteins (ERK1/2 and p38). When we compared the strength and kinetics of TCR signaling in T cell subsets, we found strikingly similar patterns of phosphoprotein activation. The maximum strength of CD4 T helper cells (CD4+ Foxp3−) signaling was greater than that of CD4 Treg (CD4+ Foxp3+ ) and CD8 T cells (CD8+ ). We also demonstrated that TCR-mediated signaling was significantly reduced in tumor-infiltrating T helper cells, but not the tumor-infiltrating CD8 cells, by measuring multiple downstream factors, as compared to normal peripheral blood T cells. These data suggest impaired T cell activation is at least partly due to failed early signaling events of TCR. doi:10.1016/j.clim.2010.03.202
T.103. Characterization of Mouse Nasal-associated Lymphoid Tissue (NALT) in BALB/c and NOD (non-obese diabetic) Mice Petra Fundová 1, Helena Tlaskalova-Hogenová 2, David Funda 2. 1Central Military Hospital, Prague, Czech Republic; 2 Institute of Microbiology, Czech Academy of Science, Prague, Czech Republic Nasal-associated lymphoid tissue (NALT) is a mucosal immune inductive site at the upper respiratory tract. It may represent an equivalent of gut(G)ALT or bronchial(B)ALT in its location and probably plays an important role in induction of immune responses after i.n. administration of antigens. NOD mouse is a well-established model in type 1 diabetes, and intranasal vaccination represents a promising way in the disease prevention. In this study, we first established an isolation protocol for preparation of cell suspensions of mouse NALT cells. Next, using FACS analysis, we analyzed the composition of NALT in SPF (specific pathogen-free) BALB/c mice and started comparisons with NOD mice. The average yield cells per NALT varied from 1.6×105 to 3.0×105. Cell viability and purity was checked by propidium iodide (PI) and CD45 staining. We evaluated basic subsets of B cells (B1 and B2) and T cells (CD4+, CD8+, and TCRγδ+) and expression of markers such as CD69 and CD62L within T (CD3-, CD4-gated) cells. Similarly, the proportion of regulatory CD4+CD25+CD45RBlow, CD4+CD103+ as