Use of a short-term whole blood intracellular staining assay to study the T-cell response in respiratory syncytial virus-infected pediatric patients

The aim of the present study was the development of a reliable method to evaluate the pattern of the ongoing T-cell response in young infants affected by respiratory infection. To this purpose, we enrolled 44 infants hospitalized with a diagnosis of respiratory syncytial virus bronchiolitis. After a short-term stimulation of whole blood samples, intracellular IFN-g and IL-4 cytokines were measured in CD4+ and CD8+ T-cell subsets by flow cytometry. A stringent staining and gating strategy was used in order to maximize the reduction of background noise and to exclude false positives. The frequencies of cytokine-producing T-cell subsets, albeit low, were easily quantifiable. Cytokine responses were higher in infants sampled > 7 days from the onset of symptoms. The use of a rigorous strategy for cell staining and gating, coupled with a short-term stimulation of whole blood and a careful evaluation of time elapsed from the onset of symptoms constitutes a convincing approach for future clinical studies.