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Phenotype And Function Of Dendritic Cells Derived From Breast Cancer Patients
AB14 Abstracts
SATURDAY
52
Phenotype And Function Of Dendritic Cells Derived From Breast Cancer Patients A. Y. Hancharou1, L. P. Titov1, L. A. Putyrski2, Y. L. Putyrski2, L. M. DuBuske3; 1Research Institute for Epidemiology and Microbiology, Minsk, BELARUS, 2Belorussian Cancer Center of N.N. Alexandrov, Minsk, BELARUS, 3Immunology Research Institute of New England, Gardner, MA. RATIONALE: Dendritic cell (DC) based therapy has been used for patients with cancer. Impaired DC function and inability of commonly used methods of induction of DC maturation to activate DC may impair therapeutic efficacy. This study characterizes monocyte-derived DC in breast cancer (BC) patients to assess their possible use as immune therapy. METHODS: Peripheral blood was obtained from 6 patients with advanced BC and 7 healthy blood donors (control group, C). Monocytes isolated from blood were cultured for 6 days in medium with GM-CSF/IL-4 to obtain immature DC (iDC). iDC were then cultured with TNF-a and dibutyryl cAMP for 24 hours to produce mature DC (mDC). mDC and allogeneic T-cells were co-cultured for 5 days to assess the stimulatory activity of mdDC on proliferation and cytokine production. Expression of CD antigens was assessed. RESULTS: In BC patients, the expression of CD1a on iDC was reduced (p50.007), while expression of CD14 was increased (p50.010) compared to C indicative of impaired response of BC DC to the cytokines used for DC differentiation. Allogeneic T-cells cultured with BC DC produced INF-g and IL-4 comparable to C. Proliferation of T-cells induced by BC DC was also comparable with C (p50.477). Expression of the co-stimulatory molecule CD80 on BC mDC was reduced (p50.004), suggesting impaired maturation of DC. However, expression of CD40, CD86, CD83 and HLADR was not impaired (p>0.05). CONCLUSIONS: Impairment of DC differentiation occurs in BC patients. There was impaired maturation of BC DC, but the functional capacities of BC DC were comparable to the control group.
53
Induction Of Innate Immune And Stress Responses By Chemicals: Role Of Toll-like Receptors, Inflammasome And Oxidative Stress In Allergic Contact Dermatitis P. R. Esser, F. C. Weber, T. M€uller, M. Idzko, T. Jakob, S. F. Martin; University Medical Center Freiburg, Freiburg, GERMANY. RATIONALE: Understanding the mechanisms of the innate immune and stress responses in the sensitization phase of allergic contact dermatitis is a crucial pre-requisite for the development of reliable in vitro test systems for the identification of chemicals with skin sensitizing potential. Moreover, interference with these responses will help to prevent the inflammation that is crucial for the sensitization to contact allergens and should therefore result in improved therapies for allergic contact dermatitis. METHODS: The role of different pattern recognition receptors, the Tolllike receptors (TLR) and NOD-like receptors (NLR) and of reactive oxygen species (ROS) was analysed in the mouse contact hypersensitivity (CHS) model. Generation and function of endogenous ligands for these receptors known to be generated in vivo during inflammatory responses was analysed in vivo and in vitro. The production of ROS and subsequent degradation of hyaluronic acid (HA) was examined. RESULTS: We provide evidence for the indirect activation of TLR2, TLR4 and the inflammasome by production of endogenous ligands in the inflammatory skin milieu triggered by contact allergens. Moreover we demonstrate a role for ROS production after contact allergen stimulation and the generation of breakdown products of extracellular matrix components like HA which serve as endogenous danger signals. CONCLUSIONS: Contact allergens can induce innate immune receptor signaling indirectly by triggering the production and/or release of endogenous danger signals in the skin microenvironment. Our findings demonstrate analogies between innate immune and stress responses to contact allergens and infections.
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
54
Dyskeratosis Congenita (DC) Presenting as Common Variable Immunodeficiency (CVID) D. Jeong1,2, S. Skoda-Smith2, A. Shimamura2, H. Ochs2; 1University of Washington, Seattle, WA, 2Seattle Children’s Hospital, Seattle, WA. RATIONALE: DC is a rare disorder characterized by marrow failure, immunologic abnormalities, malignancies, and other somatic abnormalities. Mutations in several genes are implicated, including DKC1, TERC, TERT, TINF2, NOP10, and NHP2. These mutations lead to abnormal telomere maintenance, causing shortened telomere length and impaired cell growth/proliferation. Patients with an X-linked inheritance pattern have mutations in the DKC1 gene, involved in RNA processing and telomerase function. We present the case of a 22 y/o male diagnosed with CVID in childhood, characterized by severe antibody deficiency, and DC as an adult. METHODS: Telomere length was measured in lymphocytes, granulocytes, CD45RA+/CD20- cells, CD45RA- cells, CD20+ cells, and CD57+ cells. PCR was performed for analysis of exons 1-15 of the DKC1 gene and their flanking splice sites. RESULTS: Despite IVIG and antibiotic prophylaxis, this patient developed chronic lung disease in childhood. At age 19y, he presented with refractory anemia requiring chronic transfusions, persistent diarrhea, leukoplakia, esophageal stricture, osteopenia, and oral lesions. DC was considered given his clinical history and an adolescent brother with a history of Hodgkin’s disease. Telomere length analysis revealed <1st percentile length among lymphocytes, CD45RA+/CD20- and CD57+ cells. Gene analysis revealed a c.1512_1514dupGAA mutation in the DKC1 gene, never reported before as a mutation or a benign polymorphism. He was diagnosed with likely DC. Oxymetholone caused liver toxicity and was discontinued after several doses. He passed away while undergoing bone marrow transplant. CONCLUSIONS: We present a patient with CVID and a subsequent diagnosis of DC likely caused by a novel mutation in the DKC1 gene.
55
Calcium-activated Potassium Channel KCa3.1 in the Migration and Activation of Lung Dendritic cells SHAO Z.1, D. K. Agrawal2; 1Creighton University School of Medicine, OMAHA, NE, 2Center for Clinical and Translational Science, Creighton University School of Medicine, OMAHA, NE. RATIONALE: Migration of dendritic cells (DCs) to draining lymph nodes is a critical requirement to control T cell-mediated immunity. However, the underlying mechanisms are unknown. The calcium-activated potassium channel, KCa3.1, has been shown to be involved in regulating cell migration in multiple cell types. Here, we examined the expression and the role of KCa3.1 in lung DCs. METHODS: AlexaFluor647-conjugated OVA antigen was intranasally delivered into mouse lung to label antigen-carrying DCs. Lung CD11chighCD11blow and CD11clowCD11bhigh DCs from PBS-treated and OVA-sensitized Balb/c mice were sorted using MACS and FACS. The mRNA and protein expression of KCa3.1 in lung DCs was examined using real-time PCR and flow cytometry, respectively. CCR7 expression and OVA-antigen uptake activity was measured using flow cytometry. Migration of the two lung DC subsets to lymphatic chemokines was examined in vitro using a TransWell assay in the presence and absence of the KCa3.1 blocker, TRAM-34. RESULTS: Both CD11chighCD11blow and CD11clowCD11bhigh DCs in the lung expressed KCa3.1 mRNA and protein. OVA sensitization and challenge upregulated the expression of KCa3.1 in both DC subsets with greater effect in immunogenic CD11clowCD11bhigh subset. The expression of KCa3.1 in DCs correlated well with CCR7 expression and the uptake of AlexaFluor647-conjugated OVA antigen. Blockade of KCa3.1 in vitro by TRAM-34 impaired the chemotactic migration of both lung DC subsets to lymphatic chemokines, CCL19 and CCL21. CONCLUSIONS: KCa3.1 is expressed by lung DCs and is positively associated with DC activation by OVA antigen. KCa3.1 is required for lung DC migration to lymphatic chemokines.
SATURDAY
52
Phenotype And Function Of Dendritic Cells Derived From Breast Cancer Patients A. Y. Hancharou1, L. P. Titov1, L. A. Putyrski2, Y. L. Putyrski2, L. M. DuBuske3; 1Research Institute for Epidemiology and Microbiology, Minsk, BELARUS, 2Belorussian Cancer Center of N.N. Alexandrov, Minsk, BELARUS, 3Immunology Research Institute of New England, Gardner, MA. RATIONALE: Dendritic cell (DC) based therapy has been used for patients with cancer. Impaired DC function and inability of commonly used methods of induction of DC maturation to activate DC may impair therapeutic efficacy. This study characterizes monocyte-derived DC in breast cancer (BC) patients to assess their possible use as immune therapy. METHODS: Peripheral blood was obtained from 6 patients with advanced BC and 7 healthy blood donors (control group, C). Monocytes isolated from blood were cultured for 6 days in medium with GM-CSF/IL-4 to obtain immature DC (iDC). iDC were then cultured with TNF-a and dibutyryl cAMP for 24 hours to produce mature DC (mDC). mDC and allogeneic T-cells were co-cultured for 5 days to assess the stimulatory activity of mdDC on proliferation and cytokine production. Expression of CD antigens was assessed. RESULTS: In BC patients, the expression of CD1a on iDC was reduced (p50.007), while expression of CD14 was increased (p50.010) compared to C indicative of impaired response of BC DC to the cytokines used for DC differentiation. Allogeneic T-cells cultured with BC DC produced INF-g and IL-4 comparable to C. Proliferation of T-cells induced by BC DC was also comparable with C (p50.477). Expression of the co-stimulatory molecule CD80 on BC mDC was reduced (p50.004), suggesting impaired maturation of DC. However, expression of CD40, CD86, CD83 and HLADR was not impaired (p>0.05). CONCLUSIONS: Impairment of DC differentiation occurs in BC patients. There was impaired maturation of BC DC, but the functional capacities of BC DC were comparable to the control group.
53
Induction Of Innate Immune And Stress Responses By Chemicals: Role Of Toll-like Receptors, Inflammasome And Oxidative Stress In Allergic Contact Dermatitis P. R. Esser, F. C. Weber, T. M€uller, M. Idzko, T. Jakob, S. F. Martin; University Medical Center Freiburg, Freiburg, GERMANY. RATIONALE: Understanding the mechanisms of the innate immune and stress responses in the sensitization phase of allergic contact dermatitis is a crucial pre-requisite for the development of reliable in vitro test systems for the identification of chemicals with skin sensitizing potential. Moreover, interference with these responses will help to prevent the inflammation that is crucial for the sensitization to contact allergens and should therefore result in improved therapies for allergic contact dermatitis. METHODS: The role of different pattern recognition receptors, the Tolllike receptors (TLR) and NOD-like receptors (NLR) and of reactive oxygen species (ROS) was analysed in the mouse contact hypersensitivity (CHS) model. Generation and function of endogenous ligands for these receptors known to be generated in vivo during inflammatory responses was analysed in vivo and in vitro. The production of ROS and subsequent degradation of hyaluronic acid (HA) was examined. RESULTS: We provide evidence for the indirect activation of TLR2, TLR4 and the inflammasome by production of endogenous ligands in the inflammatory skin milieu triggered by contact allergens. Moreover we demonstrate a role for ROS production after contact allergen stimulation and the generation of breakdown products of extracellular matrix components like HA which serve as endogenous danger signals. CONCLUSIONS: Contact allergens can induce innate immune receptor signaling indirectly by triggering the production and/or release of endogenous danger signals in the skin microenvironment. Our findings demonstrate analogies between innate immune and stress responses to contact allergens and infections.
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
54
Dyskeratosis Congenita (DC) Presenting as Common Variable Immunodeficiency (CVID) D. Jeong1,2, S. Skoda-Smith2, A. Shimamura2, H. Ochs2; 1University of Washington, Seattle, WA, 2Seattle Children’s Hospital, Seattle, WA. RATIONALE: DC is a rare disorder characterized by marrow failure, immunologic abnormalities, malignancies, and other somatic abnormalities. Mutations in several genes are implicated, including DKC1, TERC, TERT, TINF2, NOP10, and NHP2. These mutations lead to abnormal telomere maintenance, causing shortened telomere length and impaired cell growth/proliferation. Patients with an X-linked inheritance pattern have mutations in the DKC1 gene, involved in RNA processing and telomerase function. We present the case of a 22 y/o male diagnosed with CVID in childhood, characterized by severe antibody deficiency, and DC as an adult. METHODS: Telomere length was measured in lymphocytes, granulocytes, CD45RA+/CD20- cells, CD45RA- cells, CD20+ cells, and CD57+ cells. PCR was performed for analysis of exons 1-15 of the DKC1 gene and their flanking splice sites. RESULTS: Despite IVIG and antibiotic prophylaxis, this patient developed chronic lung disease in childhood. At age 19y, he presented with refractory anemia requiring chronic transfusions, persistent diarrhea, leukoplakia, esophageal stricture, osteopenia, and oral lesions. DC was considered given his clinical history and an adolescent brother with a history of Hodgkin’s disease. Telomere length analysis revealed <1st percentile length among lymphocytes, CD45RA+/CD20- and CD57+ cells. Gene analysis revealed a c.1512_1514dupGAA mutation in the DKC1 gene, never reported before as a mutation or a benign polymorphism. He was diagnosed with likely DC. Oxymetholone caused liver toxicity and was discontinued after several doses. He passed away while undergoing bone marrow transplant. CONCLUSIONS: We present a patient with CVID and a subsequent diagnosis of DC likely caused by a novel mutation in the DKC1 gene.
55
Calcium-activated Potassium Channel KCa3.1 in the Migration and Activation of Lung Dendritic cells SHAO Z.1, D. K. Agrawal2; 1Creighton University School of Medicine, OMAHA, NE, 2Center for Clinical and Translational Science, Creighton University School of Medicine, OMAHA, NE. RATIONALE: Migration of dendritic cells (DCs) to draining lymph nodes is a critical requirement to control T cell-mediated immunity. However, the underlying mechanisms are unknown. The calcium-activated potassium channel, KCa3.1, has been shown to be involved in regulating cell migration in multiple cell types. Here, we examined the expression and the role of KCa3.1 in lung DCs. METHODS: AlexaFluor647-conjugated OVA antigen was intranasally delivered into mouse lung to label antigen-carrying DCs. Lung CD11chighCD11blow and CD11clowCD11bhigh DCs from PBS-treated and OVA-sensitized Balb/c mice were sorted using MACS and FACS. The mRNA and protein expression of KCa3.1 in lung DCs was examined using real-time PCR and flow cytometry, respectively. CCR7 expression and OVA-antigen uptake activity was measured using flow cytometry. Migration of the two lung DC subsets to lymphatic chemokines was examined in vitro using a TransWell assay in the presence and absence of the KCa3.1 blocker, TRAM-34. RESULTS: Both CD11chighCD11blow and CD11clowCD11bhigh DCs in the lung expressed KCa3.1 mRNA and protein. OVA sensitization and challenge upregulated the expression of KCa3.1 in both DC subsets with greater effect in immunogenic CD11clowCD11bhigh subset. The expression of KCa3.1 in DCs correlated well with CCR7 expression and the uptake of AlexaFluor647-conjugated OVA antigen. Blockade of KCa3.1 in vitro by TRAM-34 impaired the chemotactic migration of both lung DC subsets to lymphatic chemokines, CCL19 and CCL21. CONCLUSIONS: KCa3.1 is expressed by lung DCs and is positively associated with DC activation by OVA antigen. KCa3.1 is required for lung DC migration to lymphatic chemokines.