Enumeration of Functional T-Cell Subsets by Fluorescence-Immunospot Defines Signatures of Pathogen Burden in Tuberculosis

Abstract

BackgroundIFN-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.MethodsWe 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.ResultsFluorescence-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.ConclusionsFluorescence-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.

A mycolic acid–specific CD1-restricted T cell population contributes to acute and memory immune responses in human tuberculosis infection

“…The relationship between antigen load and group 1 CD1-restricted T cell responses in TB is poorly understood. However, a quantitative evaluation of this relationship is essential in order to understand the role of such unconventional T cells in host defence and protective immunity and has generated important insights into the role of peptide-specific T cell subsets in TB (46)(47)(48)(49). We found that lipid-specific T cells, in common with peptide-specific T cells, have a close relationship with mycobacterial load in vivo.…”

Immunodiagnosis of Tuberculosis: a Dynamic View of Biomarker Discovery

“…One area of research has been the characterization of memory phenotypes by measuring production of IFN-␥ and IL-2, given that effector cells secrete predominantly IFN-␥, effector-memory cells secrete both IFN-␥ and IL-2, and central memory cells are known to secrete only IL-2 (99). In active TB, most antigen-specific T cells are effector cells, while central memory cells predominate in LTBI (16,129). Antibiotic treatment causes a relative decrease of cells expressing effector phenotypes and a concurrent increase of memory cells in treated TB patients (16,99).…”

Advances in tuberculosis

“…T cells secreting only TNF have recently been proposed as indicators for active TB (Harari et al, 2011). T cells secreting only IFN  were also reported to be more frequent in patients with ongoing disease (Casey et al, 2010).…”