title: Differentiation of human alloreactive CD4+ and CD8+ T cells in vitro.
abstract: BACKGROUND
  Recent studies have revealed that, during viral infection, ordered phenotypic and functional changes occur in human antigen-specific T cells. We analyzed whether a similar differentiation program is induced after alloantigen stimulation in vitro.
  
  
  METHODS
  Peripheral blood mononuclear cells and purified CD4(+)CD45RA+, CD4(+)CD45RO+, and CD8+ T cells from healthy individuals were labeled with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells were co-cultured with allogeneic irradiated cells. Flow cytometric analysis was performed on days 3, 5, and 7 of culture using surface CD45RA, CD27, CD28, CCR7, and intracellular perforin and granzyme B markers in relation to CFSE dilution.
  
  
  RESULTS
  Based on the decrease in CFSE fluorescence, both CD4+ and CD8+ T cells showed an early and vigorous response to allogeneic stimulation. Loss of CD45RA expression and up-regulation of CD27 and CD28 costimulatory molecules was an early event occurring in the first generations of dividing cells. Differentiation at later stages of proliferation was characterized by loss of CCR7 homing receptor expression, more pronounced in CD4+ than in CD8+ T cells, indicating the decreased ability of these cells to traffic to secondary lymphoid organs. Production of the cytotoxic effector molecules perforin and granzyme B increased with the number of cell divisions.
  
  
  CONCLUSIONS
  Our data thus show that short-term phenotypic and functional changes of alloreactive T cells follow the differentiation pattern seen in the early stages of an antiviral immune response.
authors.affiliation: Laboratory for Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Natalia
authors.initials: N
authors.lastname: Nikolaeva
authors.forename: Elena
authors.initials: E
authors.lastname: Uss
authors.forename: Ester M M
authors.initials: EM
authors.lastname: van Leeuwen
authors.forename: René A W
authors.initials: RA
authors.lastname: van Lier
authors.forename: Ineke J M
authors.initials: IJ
authors.lastname: ten Berge
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000276:immunology; D002469:Cell Separation / Q000379:methods; D018920:Coculture Techniques / Q000379:methods; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006084:Graft Rejection / Q000276:immunology; D006801:Humans; D008213:Lymphocyte Activation; D013388:Succinimides; D014184:Transplantation, Homologous / Q000276:immunology
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D015703:Antigens, CD; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides