title: Induction of cytolytic T lymphocytes against pediatric solid tumors in vitro using autologous dendritic cells pulsed with necrotic primary tumor.
abstract: OBJECTIVE
  Effective and generally applicable methods for generating cancer vaccines in children have not been defined. Dendritic cells (DCs) are the most potent professional antigen-presenting cells capable of activating primary cytolytic T cells. We tested the ability of DCs generated from pediatric patients' peripheral blood monocytes and pulsed with a necrotic tumor to activate autologous tumor-specific cytolytic T cells.
  
  
  METHODS
  Tumor and peripheral blood cells were obtained from pediatric patients undergoing biopsy or resection for advanced solid tumors according to an institutional research board-approved protocol and after acquiring informed consent from them. To generate DCs, we treated peripheral blood monocytes with granulocyte-macrophage colony stimulating factor and interleukin (IL)-4. Maturation was induced with a cytokine cocktail (CC) containing tumor necrosis factor-alpha, IL-6, IL-1beta, and prostaglandin E2. The DC phenotype was assayed using flow cytometry. Tumor necrosis was induced by exposure to UV-B irradiation (1000 mJ). Dendritic cells pulsed with a UV-B-treated primary tumor and matured with CC were used to stimulate autologous peripheral blood lymphocytes weekly. Tumor-specific cytolytic activity was assayed using 4-hour 51Cr release.
  
  
  RESULTS
  Peripheral blood monocytes isolated from pediatric patients differentiated into immature DCs (CD14-, MHCII+ [major histocompatibility complex], CD80(low), CD86(low)) in the presence of granulocyte-macrophage colony stimulating factor and IL-4. Cytokine cocktail induced maturation of DCs, as characterized by increased expressions of MHCII, CD83, CD80, and CD86. Patients' peripheral blood lymphocytes stimulated in vitro with DCs loaded with a necrotic primary tumor and matured with CC specifically lysed autologous neuroblastoma in 7 of 9 patients.
  
  
  CONCLUSIONS
  Dendritic cells generated from the peripheral blood of children with advanced solid tumors and pulsed with a necrotic primary tumor undergo maturation and effectively stimulate autologous tumor-specific cytolytic T cells in vitro. We describe a simple method for generating a vaccine capable of activating cytotoxic T cells against pediatric solid tumors that does not require the genetic identification of tumor-associated antigens.
authors.affiliation: Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Shilyansky
authors.forename: Paulette
authors.initials: P
authors.lastname: Jacobs
authors.forename: Kara
authors.initials: K
authors.lastname: Doffek
authors.forename: Sonia L
authors.initials: SL
authors.lastname: Sugg
mesh_terms: D000293:Adolescent; D019496:Cancer Vaccines / Q000276:immunology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002648:Child; D002675:Child, Preschool; D003713:Dendritic Cells / Q000276:immunology*; D006801:Humans; D007223:Infant; D008213:Lymphocyte Activation; D009336:Necrosis; D009369:Neoplasms / Q000276:immunology / Q000628:therapy*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D019496:Cancer Vaccines