Enumeration of functional T-cell subsets by fluorescence-immunospot defines signatures of pathogen burden in tuberculosis

Rosalyn Casey, Deena Blumenkrantz, Kerry Millington, Damien Montamat-Sicotte, Onn Min Kon, Melissa Wickremasinghe, Samuel Bremang, Murphy Magtoto, Saranya Sridhar, David Connell, Ajit Lalvani (Lead / Corresponding author)

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    Background: IFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection.

    Methods: We used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells.

    Results: Fluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells.

    Conclusions: Fluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation.

    Original languageEnglish
    Article numbere15619
    Pages (from-to)1-11
    Number of pages11
    JournalPLoS ONE
    Issue number12
    Publication statusPublished - 14 Dec 2010


    • Adolescent
    • Adult
    • Aged
    • Case-Control Studies
    • Cell Count
    • Cross-Sectional Studies
    • Female
    • Flow Cytometry/methods
    • Humans
    • Interferon-gamma/metabolism
    • Interleukin-2/metabolism
    • Male
    • Microscopy, Fluorescence/methods
    • Middle Aged
    • Mycobacterium tuberculosis/metabolism
    • Risk Factors
    • T-Lymphocytes/cytology
    • Tuberculosis/blood


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