Methods

Study population. We recruited 36 infants who were aged less than 18 months and admitted with severe RSV bronchiolitis to the “Pedro de Elizalde” Children’s Hospital in Buenos Aires, Argentina. All of these infants required hospitalization for treatment of bronchiolitis. RSV bronchiolitis was confirmed by direct immunofluorescence of nasopharyngeal aspirates. The control group consisted of 10 uninfected healthy infants admitted to the hospital for scheduled surgery. They had no identifiable airway infections for a 4-week period before the study. Characteristics of the patients are shown in Table 1. The parents of the participants enrolled in this study gave informed consent, and the study was approved by the Ethics Committee of the “Pedro de Elizalde” Hospital.

Flow cytometry. Peripheral blood mononuclear cells (PBMCs) wereisolatedfrom1mlofbloodcollectedinethylenediaminetetraacetic acid tubes through a Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Freshly isolated PBMCs were stained with anti-CD4 (APC) and anti-CD45RA (PE-Cy7) antibodies (BD Biosciences, San Jose, CA), and FOXP3 were detected in permeabilized cells with anti-FOXP3 (Alexa Fluor 488, BD Biosciences). Monocytes were excluded from the analysis based on their forward and side scatter parameters and low CD4 expression. In all cases, isotype antibodies were used as negative controls. Data were acquired using a FACSAria II (Becton Dickinson, San Jose, CA) and analyzed with FlowJo software.