title: Quantitation of dengue virus specific CD4+ T cells by intracellular cytokine staining.
abstract: We developed an intracellular cytokine staining assay to quantify dengue specific memory T cells elicited by a primary dengue virus (DEN) infection. Peripheral blood mononuclear cells (PBMC) of volunteers who received experimental live attenuated monovalent DEN vaccines were stimulated with glutaraldehyde-inactivated dengue-infected Vero cell culture lysates from all four DEN serotypes. CD4+ T cell frequencies to previously identified MHC class II peptides were equivalent to 40-70% of the responses using virus infected cell lysates in two donors. IFN-gamma responses to DEN were detected from 0.04% to 0.45% CD4+ T cells. The highest IFN-gamma response was elicited by antigens from the homologous serotype. We detected serotype-specific and -cross reactive CD4+ T cell cytokine responses from all donors. This assay is suitable for measuring DEN specific CD4+ T cells in human PBMC from large population studies where donor haplotype is unknown or highly variant.
authors.affiliation: Center for Infectious Disease and Vaccine Research, Room S5-326, 55 Lake Avenue North, University of Massachusetts Medical School, Worcester, MA 01655, USA. Marlou.Mangada@umassmed.edu
authors.forename: Marlou M
authors.initials: MM
authors.lastname: Mangada
authors.forename: Francis A
authors.initials: FA
authors.lastname: Ennis
authors.forename: Alan L
authors.initials: AL
authors.lastname: Rothman
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D004396:Coloring Agents / Q000737:chemistry; D016207:Cytokines / Q000737:chemistry / Q000276:immunology*; D003715:Dengue / Q000175:diagnosis / Q000276:immunology*; D003716:Dengue Virus / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007156:Immunologic Memory / Q000276:immunology; D007371:Interferon-gamma / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D010798:Phycocyanin / Q000737:chemistry; D014613:Vaccines, Attenuated / Q000276:immunology; D014765:Viral Vaccines / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D015704:CD4 Antigens; D004396:Coloring Agents; D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; D014613:Vaccines, Attenuated; D014765:Viral Vaccines; C002497:allophycocyanin; D010798:Phycocyanin; D007371:Interferon-gamma