title: Clonal analysis of the T-cell response to in vivo expressed Mycobacterium tuberculosis protein Rv2034, using a CD154 expression based T-cell cloning method.
abstract: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death worldwide. A better understanding of the role of CD4+ and CD8+ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of in vivo expressed Mtb genes (IVE-TB) which is expressed during in vivo pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, in vitro ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified in vivo expressed Mtb (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4+ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81-100 and Mtb lysate. Remarkably, while the recognition of the dominant p81-100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88-107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit Mtb outgrowth from infected monocytes significantly. The characterization of the polyfunctional and Mtb inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling Mtb infection, and can be applied to the analysis of T-cell responses in patient populations.
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Susanna
authors.initials: S
authors.lastname: Commandeur
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Coppola
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Karin
authors.initials: K
authors.lastname: Dijkman
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Annemieke H
authors.initials: AH
authors.lastname: Friggen
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Krista E
authors.initials: KE
authors.lastname: van Meijgaarden
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Susan J F
authors.initials: SJ
authors.lastname: van den Eeden
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Louis
authors.initials: L
authors.lastname: Wilson
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jolien J
authors.initials: JJ
authors.lastname: van der Ploeg-van Schip
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Kees L M C
authors.initials: KL
authors.lastname: Franken
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Annemieke
authors.initials: A
authors.lastname: Geluk
authors.affiliation: Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Tom H M
authors.initials: TH
authors.lastname: Ottenhoff
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D023201:CD40 Ligand / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D005260:Female; D006683:HLA-DQ Antigens / Q000276:immunology; D006684:HLA-DR Antigens / Q000276:immunology; D006801:Humans; D008297:Male; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; D006683:HLA-DQ Antigens; C051568:HLA-DQ1 antigen; D006684:HLA-DR Antigens; D012097:Repressor Proteins; C000593589:Rv2034 protein, Mycobacterium tuberculosis; D023201:CD40 Ligand