title: Selective in vitro expansion of HLA class I-restricted HIV-1 Gag-specific CD8+ T cells: cytotoxic T-lymphocyte epitopes and precursor frequencies.
abstract: OBJECTIVE
  To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor frequencies of HIV-1 Gag-specific CTL in the blood of seropositive individuals.
  
  
  METHODS
  B-lymphoblastoid cell lines (B-LCL) infected with recombinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-presenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL activity was determined in 51Cr-release assays using B-LCL as targets after infection with rVV-Gag or after pulsing with partially overlapping peptides spanning the Gag sequence.
  
  
  RESULTS
  In vitro stimulation resulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DR+ cells. Gag-specific cytotoxicity, mediated predominantly by HLA class I-restricted CD8+ CTL, was observed in all seven individuals studied. Multiple HLA-restricted CTL epitopes were identified with a single culture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine CTL precursor (CTLp) frequencies.
  
  
  CONCLUSIONS
  PFA-fixed rVV-Gag-infected autologous B-LCL can be used as stimulator cells in bulk PBMC cultures to identify CTL epitopes and to determine CTLp frequencies. This method will facilitate the analysis of HIV-1-specific CTL responses in HIV-infected and vaccinated individuals.
authors.affiliation: Laboratory of Immunobiology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
authors.forename: C A
authors.initials: CA
authors.lastname: van Baalen
authors.forename: M R
authors.initials: MR
authors.lastname: Klein
authors.forename: A M
authors.initials: AM
authors.lastname: Geretti
authors.forename: R I
authors.initials: RI
authors.lastname: Keet
authors.forename: F
authors.initials: F
authors.lastname: Miedema
authors.forename: C A
authors.initials: CA
authors.lastname: van Els
authors.forename: A D
authors.initials: AD
authors.lastname: Osterhaus
mesh_terms: D000938:Antigen-Presenting Cells / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D000939:Epitopes / Q000276:immunology; D015683:Gene Products, gag / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006680:HLA Antigens / Q000276:immunology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008213:Lymphocyte Activation; D010446:Peptide Fragments / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D000939:Epitopes; D015683:Gene Products, gag; D006680:HLA Antigens; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017493:Leukocyte Common Antigens