title: Enumeration of functional T-cell subsets by fluorescence-immunospot defines signatures of pathogen burden in tuberculosis.
abstract: 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.
authors.affiliation: Tuberculosis Research Unit, Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College London, St Mary's Hospital, London, United Kingdom.
authors.forename: Rosalyn
authors.initials: R
authors.lastname: Casey
authors.forename: Deena
authors.initials: D
authors.lastname: Blumenkrantz
authors.forename: Kerry
authors.initials: K
authors.lastname: Millington
authors.forename: Damien
authors.initials: D
authors.lastname: Montamat-Sicotte
authors.forename: Onn Min
authors.initials: OM
authors.lastname: Kon
authors.forename: Melissa
authors.initials: M
authors.lastname: Wickremasinghe
authors.forename: Samuel
authors.initials: S
authors.lastname: Bremang
authors.forename: Murphy
authors.initials: M
authors.lastname: Magtoto
authors.forename: Saranya
authors.initials: S
authors.lastname: Sridhar
authors.forename: David
authors.initials: D
authors.lastname: Connell
authors.forename: Ajit
authors.initials: A
authors.lastname: Lalvani
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D002452:Cell Count; D003430:Cross-Sectional Studies; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000378:metabolism; D012307:Risk Factors; D013601:T-Lymphocytes / Q000166:cytology*; D014376:Tuberculosis / Q000097:blood / Q000382:microbiology*
chemical_list: D007376:Interleukin-2; D007371:Interferon-gamma