Ex vivo Analysis of Cytokine Responses

Unstimulated BAL cells were stained ex vivo with anti-CD3-PE-Cy7 and CCR5-PE (both from BD) and with CD4-PE-Cy5.5 and CD8-Qdot705 (both from Invitrogen). Freshly stimulated BAL cells and stimulated cryopreserved blood cells were washed and stained with anti-CD4-PE-Cy5.5 and CD8-Qdot705 (both from BD), permeablized, and stained intracellularly with CD3-APC-H7, IFN-γ-Alexa700, and interleukin 2 (IL-2)-APC (all from BD) and with tumor necrosis factor α (TNF-α)-PE-Cy7 (eBiosciences). Cells were acquired on a BD Fortessa, using FACSDiva software, and data were analyzed using FlowJo (TreeStar) and Pestle and Spice [25 (link)]. A positive cytokine response was defined as a level that was twice the background level, a net response of >0.05%, and an event cutoff of 10 events, and all data are reported after subtraction of the background level.