Your browser doesn't support javascript.
loading
Porcine antigen-specific IFN-γ ELISpot as a potentially valuable tool for monitoring cellular immune responses in pig-to-non-human primate islet xenotransplantation.
Kim, Hyun-Je; Yoon, Il-Hee; Min, Byoung-Hoon; Kim, Yong-Hee; Shin, Jun-Seop; Kim, Jong-Min; Kim, Jung-Sik; Nam, Hye-Young; Lee, Won-Woo; Park, Chung-Gyu.
Afiliação
  • Kim HJ; Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, Korea.
  • Yoon IH; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
  • Min BH; Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea.
  • Kim YH; Department of Biomedical Sciences, Seoul National University Graduate School, Seoul, Korea.
  • Shin JS; Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, Korea.
  • Kim JM; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
  • Kim JS; Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, Korea.
  • Nam HY; Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
  • Lee WW; Institute of Endemic Diseases, Seoul National University College of Medicine, Seoul, Korea.
  • Park CG; Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, Korea.
Xenotransplantation ; 23(4): 310-9, 2016 07.
Article em En | MEDLINE | ID: mdl-27464486
BACKGROUND: Recent progress in xenotransplantation of porcine islets to non-human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen-specific cellular immune responses is necessary. The enzyme-linked immunospot (ELISpot) assay has been widely used to monitor antigen-specific alloreactive T-cell responses in humans; however, the utility of porcine islet-specific ELISpot assay has not yet been thoroughly evaluated for pig-to-NHPs intraportal islet xenotransplantation. METHODS: The optimal ELISpot assay conditions, including the number of responder and stimulator cells and the provision of costimulation, were determined. Then, ELISpot assays were conducted on serial stocks of peripheral blood mononuclear cell (PBMC) samples previously isolated from NHP recipients transplanted with porcine islets. Either splenocytes from donor pigs or pancreatic islets from third-party pigs were used for antigen stimulation. At the same time, the ratio of CD4(+) /CD8(+) T cells and the percentage of CD4(+) FoxP3(+) T cells in the peripheral blood were evaluated. Finally, liver biopsy samples were evaluated to assess the immunopathology of the grafts. RESULTS: The optimal conditions for the ELISpot assay were defined as 2.5 × 10(5) responder cells incubated with 5.0 × 10(5) stimulator cells in 96-well, flat-bottom plates without further costimulation. Using donor splenocytes as stimulators, a serial interferon-gamma (IFN-γ) ELISpot assay with PBMCs from the monkeys with prolonged porcine islet grafts (>180 days) demonstrated that the number of donor antigen-specific IFN-γ-producing cells significantly increased upon overt graft rejection. However, use of third-party porcine islets as stimulators did not reflect graft rejection, suggesting that the use of donor-specific PBMCs, and not tissue (porcine islet)-specific cells, as stimulators could better serve the purpose of this assay in adult porcine islet transplantation. IFN-γ spot number was neither influenced by the peripheral blood CD4(+) /CD8(+) T-cell ratio nor the percentage of CD4(+) FoxP3(+) T cells. Finally, in cases of overt graft rejection, the number of IFN-γ spots and the graft-infiltrating T cells in biopsied liver samples increased simultaneously. CONCLUSION: Use of PBMCs in a porcine antigen-specific IFN-γ ELISpot assay is a reliable method for monitoring T-cell-mediated rejection in pig-to-NHP islet xenotransplantation.
Assuntos
Palavras-chave

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Transplante Heterólogo / Interferon gama / Rejeição de Enxerto / Imunidade Celular Limite: Animals Idioma: En Revista: Xenotransplantation Assunto da revista: TRANSPLANTE Ano de publicação: 2016 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Transplante Heterólogo / Interferon gama / Rejeição de Enxerto / Imunidade Celular Limite: Animals Idioma: En Revista: Xenotransplantation Assunto da revista: TRANSPLANTE Ano de publicação: 2016 Tipo de documento: Article