Outline of flow cytometry staining, cellular barcoding and phosphoflow analysis.

a) Fixation, permeabilization, fluorescent barcoding and surface and intracellular staining sequence for pre- and post-tremelimumab treatment samples. b) Gating pyramid. After gating by morphology, pre- and post-dosing samples were resolved based on the Pacific orange barcoding cell labeling. c) The cells from pre- (negative for Pacific orange) and post-treatment (positive for Pacific orange) time points were then separated as CD3+ (T lymphocytes) and CD14+ (monocytes). The CD3+ cells were gated on CD4+ to analyze T helper cells, and CD4− cells to analyze for CD8+ cytotoxic T lymphocytes (CTLs). d) Intracellular phosphoproteins were analyzed in each cell subset by mean fluorescence intensity (MFI).