Tregs are absolutely required for the maintenance of self tolerance in mouse and man. Major abnormalities in Treg number or function cause rare but fatal syndromes with autoimmune, allergic and inflammatory features. Whether subtle Treg abnormalities contribute to the pathogenesis of sporadic autoimmune, allergic and immunoinflammatory diseases in man remains controversial. Robust methods for identifying and isolating human Tregs in patients and healthy controls are essential if we are to understand their role in these increasingly common diseases. We have outlined below a flow cytometric technique to detect and isolate the entire human Treg population based on expression of CD4, CD25, and CD127. Use of a number of additional antibodies for defining subsets within the Treg compartment is described. For analysis, anti-Foxp3 can be added to the cocktail, but the necessity for fixation and permeabilisation may reduce the signal from other antibodies.

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Cystic fibrosis (CF) is caused by mutations to the CF transmembrane conductance regulator (CFTR) gene. CFTR is known to be expressed on multiple immune cell subtypes, dendritic cells, monocytes/macrophages, neutrophils and lymphocytes. We hypothesized that the lack of CFTR expression on peripheral blood innate immune cells would result in an altered cell profile in the periphery and that this profile would reflect lung pathology. We performed a flow cytometric phenotypic investigation of innate immune cell proportions in peripheral blood collected from 17 CF patients and 15 age-matched healthy controls. We observed significant differences between CF patients and controls in the relative proportions of natural killer (NK) cells, monocytes and their subsets, with significant correlations observed between proportions of NK and monocyte cell subsets and lung function (forced expiratory volume in 1 sec, % predicted; FEV1% predicted) in CF patients. This study demonstrates the widespread nature of immune dysregulation in CF and provides a basis for identification of potential therapeutic targets. Modulation of the distinct CF-related immune cell phenotype identified could also be an important biomarker for evaluating CFTRtargeted drug efficacy.