title: Direct ex vivo detection of HLA-DR3-restricted cytomegalovirus- and Mycobacterium tuberculosis-specific CD4+ T cells.
abstract: In order to detect epitope-specific CD4+ T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leukocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4+ T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.84% of CD4+ T cells by intracellular cytokine staining. In peripheral blood mononuclear cells from M. tuberculosis-exposed, Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated, or CMV-seropositive individuals, we were able to directly detect with both tetramers epitope-specific T cells up to 0.62% and 0.45% of the CD4+ T-cell population reactive to M. tuberculosis and CMV, respectively. After a 6-day culture with peptide p510-522, the frequency of CMV-specific tetramer-binding T cells was expanded up to 9.90% tetramer+ CFSElow (5,6-carboxyfluorescein diacetate succinimidyl ester) cells within the CD4+ T-cell population, further confirming the specificity of the tetrameric molecules. Thus, HLA-DR3/peptide tetrameric molecules can be used to investigate HLA-DR3-restricted antigen-specific CD4+ T cells in clinical disease or after vaccination.
authors.affiliation: Department of Clinical Viro-Immunology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam.
authors.forename: Corine
authors.initials: C
authors.lastname: Bronke
authors.forename: Nanette M
authors.initials: NM
authors.lastname: Palmer
authors.forename: Geertje H A
authors.initials: GH
authors.lastname: Westerlaken
authors.forename: Mireille
authors.initials: M
authors.lastname: Toebes
authors.forename: Gijs M W
authors.initials: GM
authors.lastname: van Schijndel
authors.forename: Veenu
authors.initials: V
authors.lastname: Purwaha
authors.forename: Krista E
authors.initials: KE
authors.lastname: van Meijgaarden
authors.forename: Ton N M
authors.initials: TN
authors.lastname: Schumacher
authors.forename: Debbie
authors.initials: D
authors.lastname: van Baarle
authors.forename: Kiki
authors.initials: K
authors.lastname: Tesselaar
authors.forename: Annemieke
authors.initials: A
authors.lastname: Geluk
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002461:Cell Line, Transformed; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000175:diagnosis*; D018984:Epitopes, T-Lymphocyte; D015803:HLA-DR3 Antigen / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D014162:Transfection; D014376:Tuberculosis / Q000175:diagnosis*
chemical_list: D018984:Epitopes, T-Lymphocyte; D015803:HLA-DR3 Antigen