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339 Toll-like receptor mediated innate immune response in hepatocytes suppressed woodchuck hepatitis virus replication via interferon-independent pathway
324
Abstracts / Cytokine 43 (2008) 320–325
progressors and normal progressors have lower than normal levels of the cytokine in their sera. These data suggest that the depletion of CD4+ T cells during HIV infection results in lower levels of circulating IL-21 in HIV-infected patients. The positive correlation between numbers of CD4+ T cells and concentrations of IL-21 may suggest the use of this cytokine as a useful biomarker for the progression of HIV-infection towards AIDS. These results also suggest the potential use of this cytokine as a novel immunotherapeutic approach in HIV-infected AIDS patients. Supported by the CIHR. doi:10.1016/j.cyto.2008.07.422
339 Toll-like receptor mediated innate immune response in hepatocytes suppressed woodchuck hepatitis virus replication via interferon-independent pathway Xiaoyong Zhang 1,3, Zhongji Meng 1, Yang Xu 1,3, Jörg Schlaak 2, Michael Roggendorf 1, Mengji Lu 1, 1 Institute of Virology, University Hospital of Essen, Essen, Germany, 2 Department of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany, 3 Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Pathogen invasion may be sensed through various pattern recognition receptors (PRRs) including toll like receptors (TLRs) and helicases, that are expressed in immune cells as well as in somatic cells like hepatocytes. Previous studies have shown that TLR agonists are able to activate nonparenchymal liver cells and trigger the production of IFN to inhibit Hepatitis B virus (HBV) replication in vitro and in vivo in the transgenic mouse model. However, little is known of the PRR-mediated antiviral responses in hepatocytes. Primary hepatocytes derived from woodchuck (PWH) or murine were stimulated directly or transfected with different PRR agonists. Realtime RT-PCR showed hepatocytes cellular genes (MxA, IP10, TNF-a, IL-1b) expression were induced markedly by Pam3cys, Poly I:C, LPS and 3pRNA, which are agonists for TLR2, TLR3, TLR4, and RIG-I, respectively. Agonists for TLR8 and TLR9, R848 and CpG, were less effective, presumably due to the low expression levels of the corresponding TLRs in hepatocytes. PWHs isolated from chronic woodchuck hepatitis Virus (WHV) carrier were naturally, persistently infected with WHV. Southern blot analysis showed that only Pam3cys and LPS stimulation led to a pronounced inhibition of WHV replication in PWH. Western blot analysis showed that MAPK-ERK and PI-3K-Akt pathway were activated by Pam3cys and LPS. Consistently, U0126, a specific MAPK-ERK pathway inhibitor and PI-3k pathway inhibitor Rapamycin, but not an IFN pathway inhibitor AG490 or JNK pathway inhibitor SP600125 could block the antiviral effect of Pam3cys and LPS. Though Poly I:C and 3pRNA transfection resulted in the production of high amounts of IFN and an increased expression of cellular antiviral genes (MxA, IP10, IFNb, TNF-a, IL-1b, and IL6) over hundreds fold, no significant inhibitory effect on the WHV replication was observed. In conclusion, there are likely IFN-independent antiviral pathways leading to suppression on WHV. An activation of these pathways (ERK, PI-3K) in hepatocytes through TLRs may be useful for therapies of chronic HBV infection. doi:10.1016/j.cyto.2008.07.423
340 Effects of interferon-a on regulatory T-cell depletion in cancer immunotherapy Shawna Wall, Duane Jeansonne, Pei-Yi Lin, Aijie Liu, Gaby Rennebeck, Suzanne Thibodeaux, Carolina Livi, Ben Daniel, Tyler Curiel, Department of Medicine, Cancer Therapy & Research Center at the University of Texas Health Science Center, San Antonio, TX 78212, USA Cancer immunotherapy is hampered by immune dysfunction in the tumor microenvironment. Regulatory CD4+CD25+Foxp3+ T cells (Tregs) significantly contribute to tumorrelated immune dysfunction and defeat immunotherapy strategies. Treg depletion improves tumor immunotherapy, but is incompletely effective. The fusion toxin denileukin diftitox (ONTAK) depletes murine and human Tregs in vivo. We show that BL6 mice bearing intraperitoneal ID8 epithelial ovarian carcinoma generate dysfunctional Tregs. Intraperitoneal denileukin diftitox treatment (5 lg/mouse/week) significantly reduced Treg function and prolonged survival. We hypothesized that interferon-a would augment denileukin diftitox treatment effects. Groups of 10 BL6 mice were challenged with intraperitoneal ID8 tumor. Two weeks later, mice were treated with weekly denileukin diftitox, weekly recombinant human interferon-a (20,000 units/mouse/day 4 days of 7) or denileukin diftitox plus recombinant human interferon-a. Median survival was 81.5 days for denileukin diftitox, 85.5 days for recombinant human interferona and 107.5 days for combination therapy. Recombinant human interferon-a alone did not affect Treg function, but did boost IL-2, IL-10, IL-17, IL-22 and interferon-c from CD8+ T cells preferentially over CD4+ T cells. The mechanism for interferon-amediated enhancement of denileukin diftitox in this mouse model is under investigation. In a human patient with advanced, refractory ovarian cancer, denileukin diftitox alone reduced Tregs 15%, with no clinical response. Addition of pegylated interferon-a to denileukin diftitox effected 44% Treg depletion, associated with a positive clinical response. There was no significant effect on IL-17 production from CD4+ or CD8+ T cells. These data suggest that type I interferons could find a place in the therapeutic
armamentarium of solid tumor treatment, where efficacy thus far has been modest. One potential mechanism is to prevent regeneration of depleted Tregs. doi:10.1016/j.cyto.2008.07.424
341 Cell-associated IL-15 is the mediator of tumor immunity in B16ALPHA vaccine cells Tzu G. Wu, Theresa K. Umhoefer, Jade Cao, Robert J. Burgmeier, Christine F. Grewe, Michael R. DeWall, Alexander T. Burton, Michael J. Gerger, Molly C. Schwab, W. Robert Fleischmann Jr., The University of Minnesota Medical School, Minneapolis, MN, USA B16 melanoma cells treated for 2 weeks with IFN-alpha become B16alpha vaccine cells. Four vaccinations with lethally irradiated B16alpha vaccine cells protect 50% of mice from subsequent challenge with live, parental B16 cells. B16alpha vaccine cells accumulate (but do not secrete) significant levels of IL-15, suggesting that cell-associated IL-15 is the mediator of tumor immunity. In a deletion study, B16alpha vaccine cells were stably transfected with siRNA specific for IL-15 or with a small scrambled sequence of RNA (scRNA). B16alpha vaccine cells transfected with siRNA lost their ability to accumulate cell-associated IL-15 as well as their ability to induce tumor immunity (0% protection of mice). B16alpha vaccine cells transfected with scRNA expressed the expected amount of IL-15 and were able to induce tumor immunity (50% protection of mice). In an addition study, B16 cells were stably transfected with a gene for IL-15 (B16-IL-15 cells) that leads to the cell-associated accumulation of IL15, as shown by ELISA. Data at Day 54 post-challenge, indicate that four vaccinations with lethally irradiated B16-IL-15 cells protected 40% of mice from challenge. Vaccination with lethally irradiated B16 cells transfected with only the empty vector did not provide protection. These results definitively prove that it is the presence of cell-associated IL-15 in B16alpha vaccine cells that induces tumor immunity. Moreover, the results show the way to the development of cancer vaccines through transfection with the IL-15 gene that leads to the cell-associated accumulation of IL-15. It is envisioned that the lethally irradiated cells bearing cell-associated IL-15 release their IL-15 as they lyse within 1 or 2 days after inoculation. This released IL-15 is hypothesized to activate anergic tumor infiltrating lymphocytes that are specific for tumor antigen to break their tolerance and attack the tumor antigen bearing cells, establishing a potent antitumor activity. doi:10.1016/j.cyto.2008.07.425
342 The impact of IFNa2b on signaling of STAT1, STAT3, STAT5a, STAT5b, and SOCS3 in clinical melanoma precursors, the atypical NEVI Wenjun Wang 1, Ling Chen 1, Arash Radfar 2, John M. Kirkwood 1, 1 Division of Hematology and Oncology, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA, 2 Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA Atypical nevi are non-obligate potential precursors and risk markers of melanoma. Previous studies from our group have demonstrated the role of STAT3 as a progression marker, and the down-regulation of STAT3 and up-regulation of STAT1 by IFNa2b in melanoma and its precursors in vivo. Here we have probed STAT5a (a tumor suppressor gene) and STAT5b (an oncogene) in tumor and nevus specimens obtained from patients with melanoma and/or biopsies of nevi taken prior to and following treatment with IFNa2b (IRB-approved protocols UPCI 96-043 and UPCI 95071). STAT1 expression was found to be increased and STAT3 decreased in association with the maturation of nevocytic melanocytes in vivo. STAT5b and SOCS3 have expression patterns that follow that of STAT3 in nevi. STAT5b and SOCS3 were found to be co-localized in cultured melanocytes. STAT5a and STAT5b are expressed reciprocally in nevi and melanoma in vivo. The expression of STAT5a was associated with lesser atypia of nevi, while STAT5b was associated with greater atypia of nevi obtained pretreatment from the same individuals. Expression of STAT5b is less than that of STAT5a in nevi, while expression of STAT5b is higher in melanoma than in nevi in vivo. IFNa2b up-regulated STAT5a in 5/7 cases, and down-regulated STAT5b in 2/7 cases, while the balance of the cases revealed STAT5b to be unchanged post-treatment. To date, no effects of IFNa2b upon SOCS3 have been observed in melanocytic nevi in vivo. We conclude that IFNa2b down-regulates STAT3 and up-regulates STAT5a and STAT1 in atypical nevi of patients treated with IFNa2b in vivo. The relative expression levels of STAT5a and STAT5b are potential indices of melanocyte transformation and progression that call for larger-scale evaluation. IFNa2b may have a role in melanoma prevention through up-regulation of STAT1, STAT5a and downregulation of STAT3 and STAT5b in nevocytic melanocytes.
doi:10.1016/j.cyto.2008.07.426
Abstracts / Cytokine 43 (2008) 320–325
progressors and normal progressors have lower than normal levels of the cytokine in their sera. These data suggest that the depletion of CD4+ T cells during HIV infection results in lower levels of circulating IL-21 in HIV-infected patients. The positive correlation between numbers of CD4+ T cells and concentrations of IL-21 may suggest the use of this cytokine as a useful biomarker for the progression of HIV-infection towards AIDS. These results also suggest the potential use of this cytokine as a novel immunotherapeutic approach in HIV-infected AIDS patients. Supported by the CIHR. doi:10.1016/j.cyto.2008.07.422
339 Toll-like receptor mediated innate immune response in hepatocytes suppressed woodchuck hepatitis virus replication via interferon-independent pathway Xiaoyong Zhang 1,3, Zhongji Meng 1, Yang Xu 1,3, Jörg Schlaak 2, Michael Roggendorf 1, Mengji Lu 1, 1 Institute of Virology, University Hospital of Essen, Essen, Germany, 2 Department of Gastroenterology and Hepatology, University Hospital of Essen, Essen, Germany, 3 Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Pathogen invasion may be sensed through various pattern recognition receptors (PRRs) including toll like receptors (TLRs) and helicases, that are expressed in immune cells as well as in somatic cells like hepatocytes. Previous studies have shown that TLR agonists are able to activate nonparenchymal liver cells and trigger the production of IFN to inhibit Hepatitis B virus (HBV) replication in vitro and in vivo in the transgenic mouse model. However, little is known of the PRR-mediated antiviral responses in hepatocytes. Primary hepatocytes derived from woodchuck (PWH) or murine were stimulated directly or transfected with different PRR agonists. Realtime RT-PCR showed hepatocytes cellular genes (MxA, IP10, TNF-a, IL-1b) expression were induced markedly by Pam3cys, Poly I:C, LPS and 3pRNA, which are agonists for TLR2, TLR3, TLR4, and RIG-I, respectively. Agonists for TLR8 and TLR9, R848 and CpG, were less effective, presumably due to the low expression levels of the corresponding TLRs in hepatocytes. PWHs isolated from chronic woodchuck hepatitis Virus (WHV) carrier were naturally, persistently infected with WHV. Southern blot analysis showed that only Pam3cys and LPS stimulation led to a pronounced inhibition of WHV replication in PWH. Western blot analysis showed that MAPK-ERK and PI-3K-Akt pathway were activated by Pam3cys and LPS. Consistently, U0126, a specific MAPK-ERK pathway inhibitor and PI-3k pathway inhibitor Rapamycin, but not an IFN pathway inhibitor AG490 or JNK pathway inhibitor SP600125 could block the antiviral effect of Pam3cys and LPS. Though Poly I:C and 3pRNA transfection resulted in the production of high amounts of IFN and an increased expression of cellular antiviral genes (MxA, IP10, IFNb, TNF-a, IL-1b, and IL6) over hundreds fold, no significant inhibitory effect on the WHV replication was observed. In conclusion, there are likely IFN-independent antiviral pathways leading to suppression on WHV. An activation of these pathways (ERK, PI-3K) in hepatocytes through TLRs may be useful for therapies of chronic HBV infection. doi:10.1016/j.cyto.2008.07.423
340 Effects of interferon-a on regulatory T-cell depletion in cancer immunotherapy Shawna Wall, Duane Jeansonne, Pei-Yi Lin, Aijie Liu, Gaby Rennebeck, Suzanne Thibodeaux, Carolina Livi, Ben Daniel, Tyler Curiel, Department of Medicine, Cancer Therapy & Research Center at the University of Texas Health Science Center, San Antonio, TX 78212, USA Cancer immunotherapy is hampered by immune dysfunction in the tumor microenvironment. Regulatory CD4+CD25+Foxp3+ T cells (Tregs) significantly contribute to tumorrelated immune dysfunction and defeat immunotherapy strategies. Treg depletion improves tumor immunotherapy, but is incompletely effective. The fusion toxin denileukin diftitox (ONTAK) depletes murine and human Tregs in vivo. We show that BL6 mice bearing intraperitoneal ID8 epithelial ovarian carcinoma generate dysfunctional Tregs. Intraperitoneal denileukin diftitox treatment (5 lg/mouse/week) significantly reduced Treg function and prolonged survival. We hypothesized that interferon-a would augment denileukin diftitox treatment effects. Groups of 10 BL6 mice were challenged with intraperitoneal ID8 tumor. Two weeks later, mice were treated with weekly denileukin diftitox, weekly recombinant human interferon-a (20,000 units/mouse/day 4 days of 7) or denileukin diftitox plus recombinant human interferon-a. Median survival was 81.5 days for denileukin diftitox, 85.5 days for recombinant human interferona and 107.5 days for combination therapy. Recombinant human interferon-a alone did not affect Treg function, but did boost IL-2, IL-10, IL-17, IL-22 and interferon-c from CD8+ T cells preferentially over CD4+ T cells. The mechanism for interferon-amediated enhancement of denileukin diftitox in this mouse model is under investigation. In a human patient with advanced, refractory ovarian cancer, denileukin diftitox alone reduced Tregs 15%, with no clinical response. Addition of pegylated interferon-a to denileukin diftitox effected 44% Treg depletion, associated with a positive clinical response. There was no significant effect on IL-17 production from CD4+ or CD8+ T cells. These data suggest that type I interferons could find a place in the therapeutic
armamentarium of solid tumor treatment, where efficacy thus far has been modest. One potential mechanism is to prevent regeneration of depleted Tregs. doi:10.1016/j.cyto.2008.07.424
341 Cell-associated IL-15 is the mediator of tumor immunity in B16ALPHA vaccine cells Tzu G. Wu, Theresa K. Umhoefer, Jade Cao, Robert J. Burgmeier, Christine F. Grewe, Michael R. DeWall, Alexander T. Burton, Michael J. Gerger, Molly C. Schwab, W. Robert Fleischmann Jr., The University of Minnesota Medical School, Minneapolis, MN, USA B16 melanoma cells treated for 2 weeks with IFN-alpha become B16alpha vaccine cells. Four vaccinations with lethally irradiated B16alpha vaccine cells protect 50% of mice from subsequent challenge with live, parental B16 cells. B16alpha vaccine cells accumulate (but do not secrete) significant levels of IL-15, suggesting that cell-associated IL-15 is the mediator of tumor immunity. In a deletion study, B16alpha vaccine cells were stably transfected with siRNA specific for IL-15 or with a small scrambled sequence of RNA (scRNA). B16alpha vaccine cells transfected with siRNA lost their ability to accumulate cell-associated IL-15 as well as their ability to induce tumor immunity (0% protection of mice). B16alpha vaccine cells transfected with scRNA expressed the expected amount of IL-15 and were able to induce tumor immunity (50% protection of mice). In an addition study, B16 cells were stably transfected with a gene for IL-15 (B16-IL-15 cells) that leads to the cell-associated accumulation of IL15, as shown by ELISA. Data at Day 54 post-challenge, indicate that four vaccinations with lethally irradiated B16-IL-15 cells protected 40% of mice from challenge. Vaccination with lethally irradiated B16 cells transfected with only the empty vector did not provide protection. These results definitively prove that it is the presence of cell-associated IL-15 in B16alpha vaccine cells that induces tumor immunity. Moreover, the results show the way to the development of cancer vaccines through transfection with the IL-15 gene that leads to the cell-associated accumulation of IL-15. It is envisioned that the lethally irradiated cells bearing cell-associated IL-15 release their IL-15 as they lyse within 1 or 2 days after inoculation. This released IL-15 is hypothesized to activate anergic tumor infiltrating lymphocytes that are specific for tumor antigen to break their tolerance and attack the tumor antigen bearing cells, establishing a potent antitumor activity. doi:10.1016/j.cyto.2008.07.425
342 The impact of IFNa2b on signaling of STAT1, STAT3, STAT5a, STAT5b, and SOCS3 in clinical melanoma precursors, the atypical NEVI Wenjun Wang 1, Ling Chen 1, Arash Radfar 2, John M. Kirkwood 1, 1 Division of Hematology and Oncology, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA, 2 Department of Dermatology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA Atypical nevi are non-obligate potential precursors and risk markers of melanoma. Previous studies from our group have demonstrated the role of STAT3 as a progression marker, and the down-regulation of STAT3 and up-regulation of STAT1 by IFNa2b in melanoma and its precursors in vivo. Here we have probed STAT5a (a tumor suppressor gene) and STAT5b (an oncogene) in tumor and nevus specimens obtained from patients with melanoma and/or biopsies of nevi taken prior to and following treatment with IFNa2b (IRB-approved protocols UPCI 96-043 and UPCI 95071). STAT1 expression was found to be increased and STAT3 decreased in association with the maturation of nevocytic melanocytes in vivo. STAT5b and SOCS3 have expression patterns that follow that of STAT3 in nevi. STAT5b and SOCS3 were found to be co-localized in cultured melanocytes. STAT5a and STAT5b are expressed reciprocally in nevi and melanoma in vivo. The expression of STAT5a was associated with lesser atypia of nevi, while STAT5b was associated with greater atypia of nevi obtained pretreatment from the same individuals. Expression of STAT5b is less than that of STAT5a in nevi, while expression of STAT5b is higher in melanoma than in nevi in vivo. IFNa2b up-regulated STAT5a in 5/7 cases, and down-regulated STAT5b in 2/7 cases, while the balance of the cases revealed STAT5b to be unchanged post-treatment. To date, no effects of IFNa2b upon SOCS3 have been observed in melanocytic nevi in vivo. We conclude that IFNa2b down-regulates STAT3 and up-regulates STAT5a and STAT1 in atypical nevi of patients treated with IFNa2b in vivo. The relative expression levels of STAT5a and STAT5b are potential indices of melanocyte transformation and progression that call for larger-scale evaluation. IFNa2b may have a role in melanoma prevention through up-regulation of STAT1, STAT5a and downregulation of STAT3 and STAT5b in nevocytic melanocytes.
doi:10.1016/j.cyto.2008.07.426