- Email: [email protected]
High antitumor activity of RLI, an IL15-IL15Ralpha fusion protein, in metastatic melanoma and colorectal cancer
Abstracts / Cytokine 48 (2009) 91–137 of protection and in the onset of acute rejection. To examine whether AAT affects DC maturation in vivo, skin grafts from mice transgenic for GFP (C57Bl/6 background) were transplanted into human AAT (hAAT)- or albumin-treated balb/c mice (60 mg/ kg), and DLN were removed at different time points. Samples underwent DNA extraction for genomic PCR amplification with primers specific for GFP (3, 9 and 24 h, n = 3) or RNA extraction for RT-PCR (4 and 72 h, n = 3). In vitro, bone-marrow derived DC (BMDC) or CD11c-enriched spleen DC were added hAAT (0.5 mg/ml) 1-h before stimulation with TNFa/IFNc (10 ng/ml each). Maturation markers were analyzed by RTPCR (5, 9 and 24 h) or FACS (24 h), and cytokine release was determined by Q-PlexTM chemiluminescence-based ELISA (24 h). TNFa/IFNc-stimulated DC migration assay was preformed using recombinant MCP-1/MIP-1a. DLN from hAAT-treated mice contained less CD80 and CD86 and higher levels of IL-10 transcripts at all points, compared to control animals. DLN genotyping identified uninterrupted migration of donor cells to DLN. Similarly, hAAT allowed migration in vitro. hAAT-treated stimulated DC displayed lower levels of CD40, CD80 and CD86, released less IL-6 and MCP-1 and secreted more IL-10, compared to untreated stimulated cells. These findings suggest that hAAT generates an immature, tolerogenic, migrating DC phenotype, thus facilitating the induction of graft-protective Treg cells. doi:10.1016/j.cyto.2009.07.439
PP2-062 Protection of islet allografts by in vivo introduction of an extrachromosomal plasmid expressing human alpha-1-antitrypsin Galit Shahaf, Hadas Moser, Keren Bellacen, Mark Mizrahi, Eli C. Lewis, Poster Presentation II Protection of islet allografts by in vivo introduction of an extrachromosomal plasmid expressing human alpha-1-antitrypsin Galit Shahaf, Hadas Moser, Keren Bellacen, Mark Mizrahi, Eli C. Lewis, Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel Alpha-1-antitrypsin (AAT) is the primary circulating serine–protease inhibitor, and an anti-inflammatory and tolerogenic agent during islet allograft transplantation in mice. Clinical-grade human AAT (hAAT) is purified from pooled-plasma and processed to remove infectious contaminants. Since the molecular features of AAT are altered by the manufacturing process, and since protein moieties are detected the preparation, it is of interest to establish whether transgenic hAAT is sufficient to protect islet allografts. Sustained circulating levels of hAAT have previously been achieved by hydrodynamic tail-vein injection (HDI) of a pEF-hAAT construct that contains the genomic sequence of hAAT. Here, pEF-hAAT (100 lg/animal, n = 11) or PBS (control, n = 5) were introduced to mice by HDI. Circulating hAAT was determined by ELISA. Sixteen days later, mice were rendered hyperglycemic by streptozotocin (225 mg/kg) and grafted with allogeneic islets. Circulating hAAT levels of 1–350 lg/ ml persisted for >30 days. Islet allografts were accepted in all recipient mice that expressed hAAT and rejected in control animals. Grafts from pEF-hAAT-injected mice were surrounded by a mononuclear non-invading ‘‘cuff” containing foxp3-positive cells. According to RT-PCR, grafts displayed a marked reduction in inflammatory markers (TNFa and IL-Ib P < 0.001, iNOS and MCP-1 P < 0.05, pEF-hAAT-injected compared to control mice). In related studies, mice were grafted i.p. with 0.5cm2-pieces allogeneic skin after receiving pEF-hAAT or PBS by HDI (n = 3). Out of the total CD4positive cells that were isolated from day 7 spleens, pEF-hAAT-injected mice had a 6.17% ±1.2 foxp3-positive cell population compared to 4.2% ±0.9 in control animals. In conclusion, as low as 1 lg/ml circulating hAAT are sufficient for islet allograft protection. Therefore, it is possible that clinical-grade hAAT contains AAT with impaired islet-protective activity, thus requiring dose compensation. Since elastase inhibition by the clinical preparation is intact, our findings suggest that the islet-protective attributes of AAT are protease independent.
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the effects of low levels of circulating hAAT on islet allografts and examined the inflammatory profile of these animals during a bacterial product challenge. hAAT+/ + homozygote mice exhibited circulating hAAT levels that were unaffected by age and by inflammatory stimuli (50–200 ng/ml, ages 3 weeks to 6 months, n = 40). Transgene expression was limited to lung epithelia and was absent from macrophages and islets according to immunohistochemistry, western blot analysis and RT-PCR. Mice exhibited significantly higher levels of IL-1 receptor antagonist and low levels of IL-6 and MCP-1 in serum and lung tissue (n = 3). Examination of kinase activation profile in lung lysates of mice that were introduced heat-killed Staphylococcus epidermidis intraperitoneally revealed markedly reduced kinase activation intensities (n = 3). Fourty eight hours after streptozotocin challenge (225 mg/kg), blood glucose levels in hAAT homozygote animals were 50% lower than those elicited in wild-type mice (n = 3). In an islet allograft transplantation model, grafts were accepted in homozygote mice and rejected in wild-type mice (n = 4). Accepted grafts were decorated by the previously described ‘‘cuff” of mononuclear non-invading immune cells. We conclude that as low as 50 ng/ml transgenic circulating constitutively expressed lung-derived hAAT are sufficient to generate an anti-inflammatory cytokine milieu, improve islet viability and induce islet allograft immune protection. These low levels of the transgenic product are below its physiological anti-protease inhibitory threshold, suggesting that AAT may contain anti-inflammatory activities that are independent of protease inhibition.
doi:10.1016/j.cyto.2009.07.441
PP2-064 High antitumor activity of RLI, an IL15-IL15Ralpha fusion protein, in metastatic melanoma and colorectal cancer Anne Bessard, Véronique Solé, Grégory Bouchaud, Agnès Quéméner, Yannick Jacques, Poster Presentation II High antitumor activity of RLI, an IL15-IL15Ralpha fusion protein, in metastatic melanoma and colorectal cancer Anne Bessard, Véronique Solé, Grégory Bouchaud, Agnès Quéméner , Yannick Jacques , Inserm U892, Centre de Recherche en Cancérologie Nantes/Angers, Groupe Cytokines et Récepteurs, Nantes, France, Université de Nantes, IFR26, Nantes, France Interleukin 15 (IL-15) has an important role in tumor immunosurveillance and has a contemplated use in tumor immunotherapy. We have previously engineered the fusion protein RLI, composed of the N-terminal (amino acids 1–77, sushi+) domain of IL-15Ralpha coupled via a linker to IL-15, and shown that it displayed far better efficacy than IL15 in vitro. In this report, we investigated in vivo whether RLI would be a better alternative than IL-15 and IL-2 for cancer treatment using two distinct animal models. B16F10 mouse melanoma cells were injected in C57BL/6 mice either intravenously or intrasplenically for lung or liver metastasis, respectively. HCT-116 human colorectal cancer cells were injected in the cecum of nude mice. We show that RLI has a higher efficiency than IL-15 or IL-2 to reduce lung and liver metastasis and enhance survival in the mouse B16F10 melanoma model, a result that was associated with a higher half-life in vivo. We also found that the antitumoral effect of RLI was completely abolished by in vivo depletion of NK cells using anti-asialoGM1 Ab. Moreover, RLI was also efficient to reduce by 50% tumor growth and the progression of metastasis of human colon carcinoma cells in an orthotopic nude mouse model. The fusion protein RLI has revealed strong anticancer effect in two different cancer models overcoming the limited effect of IL-15 by increasing its bioavailability and efficiency. These findings hold significant importance for the use of RLI as a potential adjuvant/therapeutic.
These authors contributed equally to the work.
doi:10.1016/j.cyto.2009.07.442 doi:10.1016/j.cyto.2009.07.440 PP2-065 SerpinB9 expression in renal tubular epithelial cells is induced by Tolllike receptor 3
PP2-063 Islet allograft survival and anti-inflammatory cytokine profile are provided by circulating transgenic lung-specific alpha-1-antitrypsin Galit Shahaf, Nathaniel DeFelice, Chad Ficek, Frida Friedman, Charles A. Dinarello, Eli C. Lewis, Poster Presentation II Islet allograft survival and anti-inflammatory cytokine profile are provided by circulating transgenic lung-specific alpha-1-antitrypsin Galit Shahaf 1, Nathaniel DeFelice 1, Chad Ficek 1, Frida Friedman 1, Charles A. Dinarello 2, Eli C. Lewis 1, 1 Ben-Gurion University of the Negev, Faculty of Health Science, Department of Clinical Biochemistry, Beer-Sheva, Israel, 2 Division of Infectious Diseases, University of Colorado Denver, Aurora, CO, USA
Kirstin M. Heutinck, Jorien Kassies, Ineke J.M. ten Berge, Jörg Hamann, Ajda T. Rowshani, Poster Presentation II SerpinB9 expression in renal tubular epithelial cells is induced by Toll-like receptor 3 Kirstin M. Heutinck 1,2, Jorien Kassies 1,2, Ineke J.M. ten Berge 2, Jörg Hamann 1, Ajda T. Rowshani 2, 1 Department of Experimental Immunology, Renal Transplant Unit, Academic Medical Center, Amsterdam, The Netherlands, 2 Department of Internal Medicine, Renal Transplant Unit, Academic Medical Center, Amsterdam, The Netherlands
Alpha-1-antitrypsin (AAT) was found to be protective for pancreatic islets during inflammatory conditions, as well as during islet allograft transplantation and in type 1 diabetes mouse models. Whether low levels of transgenic hAAT product can achieve a similar protective effect is yet to be determined. Using the previously described lungspecific surfactant-promoter driven human AAT (hAAT) transgenic mice, we studied
Serine protease inhibitor B9 (serpinB9) is a specific inhibitor of granzyme B. The latter is used by allospecific cytotoxic T lymphocytes to induce cell death in the transplanted kidney. Previously, we have shown that renal tubular epithelial cells (TECs) have elevated serpinB9 levels during rejection. We aimed to investigate how serpinB9
PP2-062 Protection of islet allografts by in vivo introduction of an extrachromosomal plasmid expressing human alpha-1-antitrypsin Galit Shahaf, Hadas Moser, Keren Bellacen, Mark Mizrahi, Eli C. Lewis, Poster Presentation II Protection of islet allografts by in vivo introduction of an extrachromosomal plasmid expressing human alpha-1-antitrypsin Galit Shahaf, Hadas Moser, Keren Bellacen, Mark Mizrahi, Eli C. Lewis, Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel Alpha-1-antitrypsin (AAT) is the primary circulating serine–protease inhibitor, and an anti-inflammatory and tolerogenic agent during islet allograft transplantation in mice. Clinical-grade human AAT (hAAT) is purified from pooled-plasma and processed to remove infectious contaminants. Since the molecular features of AAT are altered by the manufacturing process, and since protein moieties are detected the preparation, it is of interest to establish whether transgenic hAAT is sufficient to protect islet allografts. Sustained circulating levels of hAAT have previously been achieved by hydrodynamic tail-vein injection (HDI) of a pEF-hAAT construct that contains the genomic sequence of hAAT. Here, pEF-hAAT (100 lg/animal, n = 11) or PBS (control, n = 5) were introduced to mice by HDI. Circulating hAAT was determined by ELISA. Sixteen days later, mice were rendered hyperglycemic by streptozotocin (225 mg/kg) and grafted with allogeneic islets. Circulating hAAT levels of 1–350 lg/ ml persisted for >30 days. Islet allografts were accepted in all recipient mice that expressed hAAT and rejected in control animals. Grafts from pEF-hAAT-injected mice were surrounded by a mononuclear non-invading ‘‘cuff” containing foxp3-positive cells. According to RT-PCR, grafts displayed a marked reduction in inflammatory markers (TNFa and IL-Ib P < 0.001, iNOS and MCP-1 P < 0.05, pEF-hAAT-injected compared to control mice). In related studies, mice were grafted i.p. with 0.5cm2-pieces allogeneic skin after receiving pEF-hAAT or PBS by HDI (n = 3). Out of the total CD4positive cells that were isolated from day 7 spleens, pEF-hAAT-injected mice had a 6.17% ±1.2 foxp3-positive cell population compared to 4.2% ±0.9 in control animals. In conclusion, as low as 1 lg/ml circulating hAAT are sufficient for islet allograft protection. Therefore, it is possible that clinical-grade hAAT contains AAT with impaired islet-protective activity, thus requiring dose compensation. Since elastase inhibition by the clinical preparation is intact, our findings suggest that the islet-protective attributes of AAT are protease independent.
105
the effects of low levels of circulating hAAT on islet allografts and examined the inflammatory profile of these animals during a bacterial product challenge. hAAT+/ + homozygote mice exhibited circulating hAAT levels that were unaffected by age and by inflammatory stimuli (50–200 ng/ml, ages 3 weeks to 6 months, n = 40). Transgene expression was limited to lung epithelia and was absent from macrophages and islets according to immunohistochemistry, western blot analysis and RT-PCR. Mice exhibited significantly higher levels of IL-1 receptor antagonist and low levels of IL-6 and MCP-1 in serum and lung tissue (n = 3). Examination of kinase activation profile in lung lysates of mice that were introduced heat-killed Staphylococcus epidermidis intraperitoneally revealed markedly reduced kinase activation intensities (n = 3). Fourty eight hours after streptozotocin challenge (225 mg/kg), blood glucose levels in hAAT homozygote animals were 50% lower than those elicited in wild-type mice (n = 3). In an islet allograft transplantation model, grafts were accepted in homozygote mice and rejected in wild-type mice (n = 4). Accepted grafts were decorated by the previously described ‘‘cuff” of mononuclear non-invading immune cells. We conclude that as low as 50 ng/ml transgenic circulating constitutively expressed lung-derived hAAT are sufficient to generate an anti-inflammatory cytokine milieu, improve islet viability and induce islet allograft immune protection. These low levels of the transgenic product are below its physiological anti-protease inhibitory threshold, suggesting that AAT may contain anti-inflammatory activities that are independent of protease inhibition.
doi:10.1016/j.cyto.2009.07.441
PP2-064 High antitumor activity of RLI, an IL15-IL15Ralpha fusion protein, in metastatic melanoma and colorectal cancer Anne Bessard, Véronique Solé, Grégory Bouchaud, Agnès Quéméner, Yannick Jacques, Poster Presentation II High antitumor activity of RLI, an IL15-IL15Ralpha fusion protein, in metastatic melanoma and colorectal cancer Anne Bessard, Véronique Solé, Grégory Bouchaud, Agnès Quéméner , Yannick Jacques , Inserm U892, Centre de Recherche en Cancérologie Nantes/Angers, Groupe Cytokines et Récepteurs, Nantes, France, Université de Nantes, IFR26, Nantes, France Interleukin 15 (IL-15) has an important role in tumor immunosurveillance and has a contemplated use in tumor immunotherapy. We have previously engineered the fusion protein RLI, composed of the N-terminal (amino acids 1–77, sushi+) domain of IL-15Ralpha coupled via a linker to IL-15, and shown that it displayed far better efficacy than IL15 in vitro. In this report, we investigated in vivo whether RLI would be a better alternative than IL-15 and IL-2 for cancer treatment using two distinct animal models. B16F10 mouse melanoma cells were injected in C57BL/6 mice either intravenously or intrasplenically for lung or liver metastasis, respectively. HCT-116 human colorectal cancer cells were injected in the cecum of nude mice. We show that RLI has a higher efficiency than IL-15 or IL-2 to reduce lung and liver metastasis and enhance survival in the mouse B16F10 melanoma model, a result that was associated with a higher half-life in vivo. We also found that the antitumoral effect of RLI was completely abolished by in vivo depletion of NK cells using anti-asialoGM1 Ab. Moreover, RLI was also efficient to reduce by 50% tumor growth and the progression of metastasis of human colon carcinoma cells in an orthotopic nude mouse model. The fusion protein RLI has revealed strong anticancer effect in two different cancer models overcoming the limited effect of IL-15 by increasing its bioavailability and efficiency. These findings hold significant importance for the use of RLI as a potential adjuvant/therapeutic.
These authors contributed equally to the work.
doi:10.1016/j.cyto.2009.07.442 doi:10.1016/j.cyto.2009.07.440 PP2-065 SerpinB9 expression in renal tubular epithelial cells is induced by Tolllike receptor 3
PP2-063 Islet allograft survival and anti-inflammatory cytokine profile are provided by circulating transgenic lung-specific alpha-1-antitrypsin Galit Shahaf, Nathaniel DeFelice, Chad Ficek, Frida Friedman, Charles A. Dinarello, Eli C. Lewis, Poster Presentation II Islet allograft survival and anti-inflammatory cytokine profile are provided by circulating transgenic lung-specific alpha-1-antitrypsin Galit Shahaf 1, Nathaniel DeFelice 1, Chad Ficek 1, Frida Friedman 1, Charles A. Dinarello 2, Eli C. Lewis 1, 1 Ben-Gurion University of the Negev, Faculty of Health Science, Department of Clinical Biochemistry, Beer-Sheva, Israel, 2 Division of Infectious Diseases, University of Colorado Denver, Aurora, CO, USA
Kirstin M. Heutinck, Jorien Kassies, Ineke J.M. ten Berge, Jörg Hamann, Ajda T. Rowshani, Poster Presentation II SerpinB9 expression in renal tubular epithelial cells is induced by Toll-like receptor 3 Kirstin M. Heutinck 1,2, Jorien Kassies 1,2, Ineke J.M. ten Berge 2, Jörg Hamann 1, Ajda T. Rowshani 2, 1 Department of Experimental Immunology, Renal Transplant Unit, Academic Medical Center, Amsterdam, The Netherlands, 2 Department of Internal Medicine, Renal Transplant Unit, Academic Medical Center, Amsterdam, The Netherlands
Alpha-1-antitrypsin (AAT) was found to be protective for pancreatic islets during inflammatory conditions, as well as during islet allograft transplantation and in type 1 diabetes mouse models. Whether low levels of transgenic hAAT product can achieve a similar protective effect is yet to be determined. Using the previously described lungspecific surfactant-promoter driven human AAT (hAAT) transgenic mice, we studied
Serine protease inhibitor B9 (serpinB9) is a specific inhibitor of granzyme B. The latter is used by allospecific cytotoxic T lymphocytes to induce cell death in the transplanted kidney. Previously, we have shown that renal tubular epithelial cells (TECs) have elevated serpinB9 levels during rejection. We aimed to investigate how serpinB9