title: Optimization of a whole blood intracellular cytokine assay for measuring innate cell responses to mycobacteria.
abstract: Innate cells are essential for host defense against invading pathogens, and the induction and direction of adaptive immune responses to infection. We developed and optimized a flow cytometric assay that allows measurement of intracellular cytokine expression by monocytes, dendritic cells (DC) and granulocytes, as well as cellular uptake of green-fluorescent protein (GFP)-expressing mycobacteria, in very small volumes of peripheral blood. We show that innate cell stimulation resulted in increased granularity of monocytes and mDC and decreased granulocyte granularity that precluded flow cytometric discernment of granulocytes from monocytes and myeloid DC by forward and side scatter gating. Anti-CD66a/c/e antibody staining allowed reliable identification and exclusion of granulocytes for subsequent delineation of monocytes and myeloid DC. Intracellular cytokine expression by granulocytes, monocytes and mDC was remarkably sensitive to the dose of mycobacterial inoculum. Moreover, activation of monocytes and mDC with live BCG reduced expression levels of CD14 and CD11c, respectively, necessitating optimization of staining conditions to reliably measure these lineage markers. Finally, we characterized expression of IL-12/23p40, TNF-α, IL-6, and IL-10, by GFP(+) and GFP(-) monocytes and mDC from 25 healthy adults. This assay may be applied to the study of innate cell responses to any GFP-expressing pathogen, and can be performed on blood volumes as low as 200 μL per condition, making the assay particularly suitable for pediatric studies.
authors.affiliation: South African Tuberculosis Vaccine Initiative, Institute of Infectious Diseases and Molecular Medicine and School of Child and Adolescent Health, University of Cape Town, Cape Town, South Africa.
authors.forename: Muki S
authors.initials: MS
authors.lastname: Shey
authors.forename: E Jane
authors.initials: EJ
authors.lastname: Hughes
authors.forename: Marwou
authors.initials: M
authors.lastname: de Kock
authors.forename: Charlene
authors.initials: C
authors.lastname: Barnard
authors.forename: Lynnett
authors.initials: L
authors.lastname: Stone
authors.forename: Tobias R
authors.initials: TR
authors.lastname: Kollmann
authors.forename: Willem A
authors.initials: WA
authors.lastname: Hanekom
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Scriba
mesh_terms: D000293:Adolescent; D000328:Adult; D016207:Cytokines / Q000097:blood / Q000276:immunology*; D003713:Dendritic Cells / Q000276:immunology / Q000382:microbiology; D005434:Flow Cytometry / Q000379:methods*; D006098:Granulocytes / Q000276:immunology / Q000382:microbiology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007113:Immunity, Innate / Q000276:immunology; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology / Q000382:microbiology; D009161:Mycobacterium / Q000276:immunology*; D009164:Mycobacterium Infections / Q000097:blood / Q000276:immunology* / Q000382:microbiology / Q000517:prevention & control; D055815:Young Adult
chemical_list: D016207:Cytokines; D049452:Green Fluorescent Proteins