title: Quantification of antigen-reactive T cells by a modified ELISPOT assay based on freshly isolated blood dendritic cells.
abstract: The enzyme-linked immunospot (ELISPOT) assay has become a widely employed method for quantification of antigen-reactive T lymphocytes. In recent years, various types of antigen-presenting cells (APCs) have been tested as stimulator cells in ELISPOT protocols to achieve a highly sensitive and rapid assay which is not impaired by a marked nonspecific cytokine release. However, the currently available APCs still have disadvantages, such as significant background reactivities, limited sensitivity, and time-consuming preparation procedures. Recently, we succeeded in defining a novel subpopulation of circulating dendritic cells (DCs) that can easily be prepared from human blood. These M-DC8+ DCs proved to be very effective in the induction of antigen-specific T cell responses. In the present study we provide evidence that M-DC8+ DCs are particularly well suited as APCs for the detection of antigen-specific CD8+ T cells after challenge with viral or tumor peptides in ELISPOT assays. In addition, protein-loaded M-DC8+ DCs proved to be quite efficient in the presentation of MHC class II-bound peptides, thus allowing the determination of frequencies of antigen-reactive CD4+ T cells. The use of M-DC8+ DCs as stimulator cells can improve the ELISPOT assay by combining high sensitivity, rapidity, and low background reactivity.
authors.affiliation: Institute of Immunology, Medical Faculty, Technical University, Dresden, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Schmitz
authors.forename: J
authors.initials: J
authors.lastname: Rohayem
authors.forename: R
authors.initials: R
authors.lastname: Paul
authors.forename: B
authors.initials: B
authors.lastname: Weigle
authors.forename: A
authors.initials: A
authors.lastname: Stein
authors.forename: E P
authors.initials: EP
authors.lastname: Rieber
mesh_terms: D003713:Dendritic Cells / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007371:Interferon-gamma / Q000276:immunology*; D008545:Melanoma / Q000097:blood; D014442:Monophenol Monooxygenase / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D007371:Interferon-gamma; D014442:Monophenol Monooxygenase