BACKGROUND: IFN-gamma 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-gamma/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-gamma 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-gamma-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-gamma/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-gamma-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-gamma/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-gamma/IL-2-secreting T-cells and lower proportions of IFN-gamma-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.