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

• Published in: PloS one Vol. 5; no. 12; p. e15619
• Main Authors: Casey, Rosalyn, Blumenkrantz, Deena, Millington, Kerry, Montamat-Sicotte, Damien, Kon, Onn Min, Wickremasinghe, Melissa, Bremang, Samuel, Magtoto, Murphy, Sridhar, Saranya, Connell, David, Lalvani, Ajit
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.12.2010; Public Library of Science (PLoS)
• Subjects: Adolescent; Adult; Aged; Antibodies; Antigens; Biology; Case-Control Studies; Cell Count; Containment; Cross-Sectional Studies; Cytokines; Cytometry; Digital imaging; Disease; Enumeration; Enzymes; Feasibility studies; Female; Flow Cytometry - methods; Fluorescence; Health aspects; Heart; Hospitals; Humans; Immunologic factors; Infections; Interferon; Interferon-gamma - metabolism; Interleukin 2; Interleukin-2 - metabolism; Lymphocytes; Lymphocytes T; Male; Medical research; Medicine; Microscopy, Fluorescence - methods; Middle Aged; Mycobacterium tuberculosis; Mycobacterium tuberculosis - metabolism; Pathogens; Patients; Risk Factors; Signatures; T cell receptors; T cells; T-Lymphocytes - cytology; Tuberculosis; Tuberculosis - blood; Tuberculosis - microbiology; Viral infections; γ-Interferon; United Kingdom > UK
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0015619

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. 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. 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. 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.