Clonal analysis of the T-cell response to in vivo expressed Mycobacterium tuberculosis protein Rv2034, using a CD154 expression based T-cell cloning method

Susanna Commandeur, Mariateresa Coppola, Karin Dijkman, Annemieke H. Friggen, Krista E. Van Meijgaarden, Susan J.F. Van Den Eeden, Louis Wilson, Jolien J. Van Der Ploeg-Van Schip, Kees L.M.C. Franken, Annemieke Geluk, Tom H.M. Ottenhoff

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Article numbere99203
JournalPLoS ONE
Volume9
Issue number6
DOIs
Publication statusPublished - 6 Jun 2014

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