- Email: [email protected]
RNA-loaded CD40-activated B cells stimulate antigen-specific T cell responses in dogs with spontaneous lymphoma
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
341
Characterization of hematological and immunological parameters during sub clinical phase of Ehrlichia canis infection in dogs
RNA-loaded CD40-activated B cells stimulate antigenspecific T cell responses in dogs with spontaneous lymphoma
Fernanda Fidelis Gonsales Guimarães 1,∗ , Juliana Sayuri Kuribayashi 1 , Cintia Raquel Bombardieri 1 , Maristela Martins de Camargo 1 , Mitika Kuribayashi Hagiwara 2
Nicola J. Mason 1,2,3,∗ , Christina M. Coughlin 1,4 , Jarish N. Cohen 1 , Theresa A. Colligon 1 , Craig A. Clifford 6 , Andreas Zurbriggen 7 , Karin U. Sorenmo 3 , Robert H. Vonderheide 1,5
1
Instituto de Ciências Biomédicas - departamento de Imunologia da Universidade de São Paulo, Brazil 2 Faculdade de Medicina Veterinária e Zootecnia da USP departamento de clínica médica da Universidade de São Paulo, Brazil Keywords: Ehrlichia canis; Inversion of CD4+:CD8+; Dogs Species: Canine The course of infection by E. canis can be divided into 3 phases, acute, sub clinical and chronic, based on symptoms and clinical-pathologic alterations. The acute phase, ranging from 3 to 5 weeks of duration, is characterized by the development of multi-systemic symptoms due to the intense inflammatory process triggered by infection. Upon resolution of the acute phase, those dogs unable to eliminate the infectious agent become carriers. These dogs are clinically healthy, although some present variable degrees of thrombocytopenia and leucopenia. With progression of infection, these dogs develop pancytopenia with reserved prognostic. Previously it was shown that high levels of IgG, increase in CD8+ number and inversion of CD4+:CD8+ ratio develop during the acute phase of E. canis infection. Herein we analyzed the humoral response and lymphocyte subpopulations in 5 asymptomatic dogs presenting discrete leucopenia and thrombocytopenia. Four dogs have been infected for four years and one has been infected for an undetermined period of time. Antibodies were quantified by IFI and CD4+ and CD8+ lymphocytes were quantified by flow cytometry. As controls, we analyzed 8 clinically healthy dogs with no morbid history suggestive of canine ehrlichiosis and no hematological alterations. These animals were also negative for specific anti-ehrlichia antibodies and for presence of E. canis DNA (by PCR). All animals in the infected group were positive in the PCR analysis for E. canis DNA and presented anti-E. canis titles ranging from 40.960 and 81.920. Healthy individuals presented 45.7 ± 6.6% of CD4+ cells, 22.6 ± 6.1% of CD8+ cells and a ratio of 2.25 for CD4+:CD8+. E. canis-infected dogs presented 33.7 ± 10.6% of CD4+ cells, 51.6 ± 21.3% of CD8+ cells and a ratio of 0.85 for CD4+:CD8+. The high title of specific antibodies and the inversion of T CD4+:CD8+ ratio are compatible with intense activity of the immune system against the infectious agent. The hematological alterations observed in terminal canine erhliquiosis might be associated with the inflammatory and immunemediated reactions triggered by the persistence of the infection. doi:10.1016/j.vetimm.2008.10.277
1
Abramson Family Cancer Research Institute, University of Pennsylvania, School of Medicine, United States 2 Department of Pathobiology, United States 3 Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, United States 4 Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, United States 5 Hematology-Oncology Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States 6 Oncology Service, Red Bank Veterinary Hospital, Red Bank, NJ, United States 7 Institute of Animal Neurology, University of Bern, Bern, Switzerland Keywords: CD40; B cell; Cancer immunotherapy; Canine Species: Canine Cell-based vaccination strategies to induce tumorspecific T cells and generate anti-tumor immunity have largely focused on the use of autologous dendritic cells to trigger antigen-specific T cell responses in cancer patients. CD40 activated B cells (CD40-B) loaded with tumor antigen are a highly efficient alternative antigen-presenting cell capable of priming naïve T cells against neoantigens, boosting memory T cell responses and breaking tolerance to tumor-associated antigens. The use of tumor RNA as the antigenic payload for CD40-B appears especially promising and allows for gene transfer without the use of viruses or vectors and permits an MHC-independent, multiple-antigen targeting approach important in cancers where few tumor-associated antigens have been described. However, the in vivo efficacy of RNA-transfected CD40-B cells to stimulate tumor-specific T cell immunity remains immunologically and clinically untested. Development of a large animal model of spontaneous cancer to evaluate the safety and efficacy of RNA-loaded CD40-B cells is warranted prior to use of these promising alternative APCs in clinical vaccination of human cancer patients. In this study, we characterize a novel culture system consisting of CD40L transfected K562 cells (KtCD40L) to generate functional CD40-B cells from both human and canine PBMCs. Furthermore, we demonstrate that RNA-loaded CD40-B cells can induce functional antigen-specific CTLs from humans, healthy canine donors, and privately owned dogs with spontaneously occurring lymphoma. Our data demonstrate that RNA-loaded autologous CD40-B can stimulate antigenspecific T cell responses in vitro in dogs, paving the way toward the first clinical immunotherapy trial using tumor RNA-loaded CD40-B cells in vivo to stimulate anti-tumor
342
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
immunity in a large animal model of spontaneously occurring neoplasia. doi:10.1016/j.vetimm.2008.10.278 Harvest and characterization of mesenchymal canine stem cells from adipose tissue and bone marrow A.C.C. Silveira 1,∗ , R.S. Lima 2 , E.M. Penha 1 , S.G. 2 2 Macambira , M.B.P. Soares , R. Ribeiro-Dos-Santos 2 , S.M. Barrouin-Melo 1 , P.H.P. Aguiar 1 1
Escola Medicina Veterinária da UFBA, Dep. Patologia e Clínicas, Brazil 2 Centro de Pesquisas Gonc¸alo Moniz–CPqGM/FIOCRUZ, Brazil Keywords: Mesenchymal stem cells; Adipose tissue; Bone marrow Species: Canine Stem cells differ from other kinds of cells in the body. All stem cells, regardless of their source, have three general properties: (i) they are capable of dividing and renewing themselves for long periods; (ii) they are unspecialized; (iii) they can give rise to specialized cell types. The increasing approach for the use of stem cells with therapeutic purposes has been occurring due to their capacity of hematopoietic reconstruction and mainly to their plasticity, what allows their differentiation in diverse tissues, such as liver, central nervous system, skeletal muscle and others. Stem cells are present in early embryonic development and are found in mature adults. Potential therapies based on adult stem cells are appealing because their obtaining does not require destruction of the host and is therefore less ethical controversial. Therefore, adult stem cells from the bone marrow stroma and adipose tissue have been proposed as an alternative source. Adipose tissue and bone marrow are derived from the mesenchyme and contain a supportive stroma, which is easily isolated. These cells can be obtained in large numbers at high frequency from a tissue source that can be extracted in large quantities with minimal morbidity. The aim of this study was to standardize and characterize a method for obtaining mesenchymal stem cells from dog’s adipose tissue and of bone marrow. Material and methods: Eight healthy mongrel dogs were used. The bone marrow aspirative puncture was performed in the posterior iliac crest. The adipose tissue was collected by liposuction with manual cannula in the dorsal area and treated with Collagenase Sigma Type IA. Stem cells were harvested from bone marrow and adipose tissue with Ficoll Histopaque 1119 and 1077 and counted. The cells were kept under culture with DMEM medium and analysed by FACS with monoclonal antibodies specific for CD34, CD105, c-Kit, GFAP and AF Protein. Results: Each bone marrow aspirative puncture yielded an average of 7 × 107 cells/mL while each lipo-aspiration allowed an average of 1,5 × 106 cells/mL. The cells were positive for the markers CD34, CD105, c-Kit, GFAP and AFProtein.
Conclusions: These preliminary results show that the cells which were harvested from canine bone marrow and adipose tissue could be characterized as canine stem cells. The procedures were safe and allow its use as stem cell model for therapeutic purpose and scientific studies thus giving important information in human pre-clinical trials with stem cell. doi:10.1016/j.vetimm.2008.10.279 Immunological changes in dogs with chronic renal failure S. Kralova 1 , L. Leva 2 , M. Toman 1,2,∗ 1 University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic 2 Veterinary Research Institute, Brno, Czech Republic Keywords: Immunopathology; Chronic renal failure; Lymphocyte; Immunosuppression
E-mail address: [email protected] (M. Toman). Species: Canine Chronic renal failure (CRF) is the condition of decreased renal function, in which kidneys fail to maintain stability of internal environment even in basal conditions. The disease is always accompanied by azotemia characterised by elevated levels of creatinine and urea in the blood. Clinical signs are of various intensities. An adverse effect of CRF on the immune system and immunosuppression has been described in humans; however, knowledge of CRF effect on the immune system in animals is only partial. In our study on 40 dogs – clinical patients with various degrees of CRF – we investigated a set of biochemical, haematological and immunological parameters. The patients were allocated into three groups according to two criteria. First criterion was manifestation of clinical symptoms—group 1 was without clinical symptoms and with diagnosed azotemia, group 2 was with moderate clinical symptoms and group 3 was in the final stage of CRF. Second criterion was plasma creatinine level. Our study confirmed that depending on development of clinical symptoms, anaemia and lymphopoenia appeared and creatinine, urea and phosphorus plasma levels increased. Besides development of clinical symptoms and changing biochemical parameters, lymphopoenia were accompanied by a decreasing activity of lymphocytes in the test of lymphocyte transformation both in resting and mitogen stimulated cells. We did not find changes of lymphocyte subsets ratio in affected animals. We also tested the effect of serum from diseased animals on in vitro activities of lymphocytes and neutrophils. The relationship between renal failure and respective parameters of immune responses is discussed in the presentation. Supported by grant MZE 0002716201. doi:10.1016/j.vetimm.2008.10.280
341
Characterization of hematological and immunological parameters during sub clinical phase of Ehrlichia canis infection in dogs
RNA-loaded CD40-activated B cells stimulate antigenspecific T cell responses in dogs with spontaneous lymphoma
Fernanda Fidelis Gonsales Guimarães 1,∗ , Juliana Sayuri Kuribayashi 1 , Cintia Raquel Bombardieri 1 , Maristela Martins de Camargo 1 , Mitika Kuribayashi Hagiwara 2
Nicola J. Mason 1,2,3,∗ , Christina M. Coughlin 1,4 , Jarish N. Cohen 1 , Theresa A. Colligon 1 , Craig A. Clifford 6 , Andreas Zurbriggen 7 , Karin U. Sorenmo 3 , Robert H. Vonderheide 1,5
1
Instituto de Ciências Biomédicas - departamento de Imunologia da Universidade de São Paulo, Brazil 2 Faculdade de Medicina Veterinária e Zootecnia da USP departamento de clínica médica da Universidade de São Paulo, Brazil Keywords: Ehrlichia canis; Inversion of CD4+:CD8+; Dogs Species: Canine The course of infection by E. canis can be divided into 3 phases, acute, sub clinical and chronic, based on symptoms and clinical-pathologic alterations. The acute phase, ranging from 3 to 5 weeks of duration, is characterized by the development of multi-systemic symptoms due to the intense inflammatory process triggered by infection. Upon resolution of the acute phase, those dogs unable to eliminate the infectious agent become carriers. These dogs are clinically healthy, although some present variable degrees of thrombocytopenia and leucopenia. With progression of infection, these dogs develop pancytopenia with reserved prognostic. Previously it was shown that high levels of IgG, increase in CD8+ number and inversion of CD4+:CD8+ ratio develop during the acute phase of E. canis infection. Herein we analyzed the humoral response and lymphocyte subpopulations in 5 asymptomatic dogs presenting discrete leucopenia and thrombocytopenia. Four dogs have been infected for four years and one has been infected for an undetermined period of time. Antibodies were quantified by IFI and CD4+ and CD8+ lymphocytes were quantified by flow cytometry. As controls, we analyzed 8 clinically healthy dogs with no morbid history suggestive of canine ehrlichiosis and no hematological alterations. These animals were also negative for specific anti-ehrlichia antibodies and for presence of E. canis DNA (by PCR). All animals in the infected group were positive in the PCR analysis for E. canis DNA and presented anti-E. canis titles ranging from 40.960 and 81.920. Healthy individuals presented 45.7 ± 6.6% of CD4+ cells, 22.6 ± 6.1% of CD8+ cells and a ratio of 2.25 for CD4+:CD8+. E. canis-infected dogs presented 33.7 ± 10.6% of CD4+ cells, 51.6 ± 21.3% of CD8+ cells and a ratio of 0.85 for CD4+:CD8+. The high title of specific antibodies and the inversion of T CD4+:CD8+ ratio are compatible with intense activity of the immune system against the infectious agent. The hematological alterations observed in terminal canine erhliquiosis might be associated with the inflammatory and immunemediated reactions triggered by the persistence of the infection. doi:10.1016/j.vetimm.2008.10.277
1
Abramson Family Cancer Research Institute, University of Pennsylvania, School of Medicine, United States 2 Department of Pathobiology, United States 3 Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, United States 4 Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, United States 5 Hematology-Oncology Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States 6 Oncology Service, Red Bank Veterinary Hospital, Red Bank, NJ, United States 7 Institute of Animal Neurology, University of Bern, Bern, Switzerland Keywords: CD40; B cell; Cancer immunotherapy; Canine Species: Canine Cell-based vaccination strategies to induce tumorspecific T cells and generate anti-tumor immunity have largely focused on the use of autologous dendritic cells to trigger antigen-specific T cell responses in cancer patients. CD40 activated B cells (CD40-B) loaded with tumor antigen are a highly efficient alternative antigen-presenting cell capable of priming naïve T cells against neoantigens, boosting memory T cell responses and breaking tolerance to tumor-associated antigens. The use of tumor RNA as the antigenic payload for CD40-B appears especially promising and allows for gene transfer without the use of viruses or vectors and permits an MHC-independent, multiple-antigen targeting approach important in cancers where few tumor-associated antigens have been described. However, the in vivo efficacy of RNA-transfected CD40-B cells to stimulate tumor-specific T cell immunity remains immunologically and clinically untested. Development of a large animal model of spontaneous cancer to evaluate the safety and efficacy of RNA-loaded CD40-B cells is warranted prior to use of these promising alternative APCs in clinical vaccination of human cancer patients. In this study, we characterize a novel culture system consisting of CD40L transfected K562 cells (KtCD40L) to generate functional CD40-B cells from both human and canine PBMCs. Furthermore, we demonstrate that RNA-loaded CD40-B cells can induce functional antigen-specific CTLs from humans, healthy canine donors, and privately owned dogs with spontaneously occurring lymphoma. Our data demonstrate that RNA-loaded autologous CD40-B can stimulate antigenspecific T cell responses in vitro in dogs, paving the way toward the first clinical immunotherapy trial using tumor RNA-loaded CD40-B cells in vivo to stimulate anti-tumor
342
Abstracts / Veterinary Immunology and Immunopathology 128 (2009) 211–347
immunity in a large animal model of spontaneously occurring neoplasia. doi:10.1016/j.vetimm.2008.10.278 Harvest and characterization of mesenchymal canine stem cells from adipose tissue and bone marrow A.C.C. Silveira 1,∗ , R.S. Lima 2 , E.M. Penha 1 , S.G. 2 2 Macambira , M.B.P. Soares , R. Ribeiro-Dos-Santos 2 , S.M. Barrouin-Melo 1 , P.H.P. Aguiar 1 1
Escola Medicina Veterinária da UFBA, Dep. Patologia e Clínicas, Brazil 2 Centro de Pesquisas Gonc¸alo Moniz–CPqGM/FIOCRUZ, Brazil Keywords: Mesenchymal stem cells; Adipose tissue; Bone marrow Species: Canine Stem cells differ from other kinds of cells in the body. All stem cells, regardless of their source, have three general properties: (i) they are capable of dividing and renewing themselves for long periods; (ii) they are unspecialized; (iii) they can give rise to specialized cell types. The increasing approach for the use of stem cells with therapeutic purposes has been occurring due to their capacity of hematopoietic reconstruction and mainly to their plasticity, what allows their differentiation in diverse tissues, such as liver, central nervous system, skeletal muscle and others. Stem cells are present in early embryonic development and are found in mature adults. Potential therapies based on adult stem cells are appealing because their obtaining does not require destruction of the host and is therefore less ethical controversial. Therefore, adult stem cells from the bone marrow stroma and adipose tissue have been proposed as an alternative source. Adipose tissue and bone marrow are derived from the mesenchyme and contain a supportive stroma, which is easily isolated. These cells can be obtained in large numbers at high frequency from a tissue source that can be extracted in large quantities with minimal morbidity. The aim of this study was to standardize and characterize a method for obtaining mesenchymal stem cells from dog’s adipose tissue and of bone marrow. Material and methods: Eight healthy mongrel dogs were used. The bone marrow aspirative puncture was performed in the posterior iliac crest. The adipose tissue was collected by liposuction with manual cannula in the dorsal area and treated with Collagenase Sigma Type IA. Stem cells were harvested from bone marrow and adipose tissue with Ficoll Histopaque 1119 and 1077 and counted. The cells were kept under culture with DMEM medium and analysed by FACS with monoclonal antibodies specific for CD34, CD105, c-Kit, GFAP and AF Protein. Results: Each bone marrow aspirative puncture yielded an average of 7 × 107 cells/mL while each lipo-aspiration allowed an average of 1,5 × 106 cells/mL. The cells were positive for the markers CD34, CD105, c-Kit, GFAP and AFProtein.
Conclusions: These preliminary results show that the cells which were harvested from canine bone marrow and adipose tissue could be characterized as canine stem cells. The procedures were safe and allow its use as stem cell model for therapeutic purpose and scientific studies thus giving important information in human pre-clinical trials with stem cell. doi:10.1016/j.vetimm.2008.10.279 Immunological changes in dogs with chronic renal failure S. Kralova 1 , L. Leva 2 , M. Toman 1,2,∗ 1 University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic 2 Veterinary Research Institute, Brno, Czech Republic Keywords: Immunopathology; Chronic renal failure; Lymphocyte; Immunosuppression
E-mail address: [email protected] (M. Toman). Species: Canine Chronic renal failure (CRF) is the condition of decreased renal function, in which kidneys fail to maintain stability of internal environment even in basal conditions. The disease is always accompanied by azotemia characterised by elevated levels of creatinine and urea in the blood. Clinical signs are of various intensities. An adverse effect of CRF on the immune system and immunosuppression has been described in humans; however, knowledge of CRF effect on the immune system in animals is only partial. In our study on 40 dogs – clinical patients with various degrees of CRF – we investigated a set of biochemical, haematological and immunological parameters. The patients were allocated into three groups according to two criteria. First criterion was manifestation of clinical symptoms—group 1 was without clinical symptoms and with diagnosed azotemia, group 2 was with moderate clinical symptoms and group 3 was in the final stage of CRF. Second criterion was plasma creatinine level. Our study confirmed that depending on development of clinical symptoms, anaemia and lymphopoenia appeared and creatinine, urea and phosphorus plasma levels increased. Besides development of clinical symptoms and changing biochemical parameters, lymphopoenia were accompanied by a decreasing activity of lymphocytes in the test of lymphocyte transformation both in resting and mitogen stimulated cells. We did not find changes of lymphocyte subsets ratio in affected animals. We also tested the effect of serum from diseased animals on in vitro activities of lymphocytes and neutrophils. The relationship between renal failure and respective parameters of immune responses is discussed in the presentation. Supported by grant MZE 0002716201. doi:10.1016/j.vetimm.2008.10.280