title: Amplification of low-frequency antiviral CD8 T cell responses using autologous dendritic cells.
abstract: OBJECTIVE
  To utilize the potent antigen-presenting capacity of mature dendritic cells (MDC) in order to develop a rapid, sensitive method for quantifying antigen-specific CD8 T cells present at low frequency in peripheral blood.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMC) were obtained from seven HIV-1-positive individuals with low to moderate CD8 T cell responses, including five on highly active antiretroviral therapy (HAART). IFN-gamma ELISPOT assays were performed using either monocytes or MDC to present antigens expressed by recombinant vaccinia viruses (r-VV).
  
  
  METHODS
  Peripheral blood-derived monocytes were cultured for 5-6 days in the presence of IL-4 and granulocyte macrophage colony-stimulating factor, then matured in monocyte-conditioned medium. MDC were infected with r-VV and co-cultured in an ELISPOT assay with autologous monocyte-depleted PBMC.
  
  
  RESULTS
  Relative to autologous monocytes, MDC amplified detection of antigen-specific CD8 T cells by 2-30-fold in response to antigens from HIV-1, Epstein-Barr virus and cytomegalovirus. Furthermore, antigenic specificities were revealed that had not been detected using standard ELISPOT of PBMC.
  
  
  CONCLUSIONS
  This assay will prove useful for the detection of memory T cells present at low frequency, and may be of interest for identifying subdominant cytotoxic T lymphocyte epitopes. This method may have broad applications for the detection of antiviral CD8 T cell responses in patient populations in whom such responses have been difficult to detect, including HIV-1-seropositive individuals with advanced disease or undergoing HAART.
authors.affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY, USA.
authors.forename: Marie
authors.initials: M
authors.lastname: Larsson
authors.forename: David T
authors.initials: DT
authors.lastname: Wilkens
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Fonteneau
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Beadle
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Merritt
authors.forename: Rhonda G
authors.initials: RG
authors.lastname: Kost
authors.forename: Patrick A J
authors.initials: PA
authors.lastname: Haslett
authors.forename: Susan
authors.initials: S
authors.lastname: Cu-Uvin
authors.forename: Nina
authors.initials: N
authors.lastname: Bhardwaj
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Nixon
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Shacklett
mesh_terms: D000328:Adult; D017951:Antigen Presentation / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003713:Dendritic Cells / Q000276:immunology*; D005189:False Positive Reactions; D005260:Female; D005434:Flow Cytometry; D005615:Freezing; D005822:Genetic Vectors; D015658:HIV Infections / Q000188:drug therapy / Q000276:immunology* / Q000628:therapy; D015497:HIV-1 / Q000276:immunology*; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000276:immunology; D007167:Immunotherapy / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology; D014616:Vaccinia virus; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins
chemical_list: C068382:BRLF1 protein, Human herpesvirus 4; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C065418:cytomegalovirus matrix protein 65kDa