Th17 cells are detected in PB and do not significantly overlap with Th1 CD4 T cells. PB lymphocytes were stimulated with anti-CD3 overnight in the presence of brefeldin A. Cells were then stained and analyzed by flow cytometry as described in “Intracellular cytokine assay.” (A) Cells were gated based on characteristic light scatter properties, followed by positive staining for CD3 without binding to the dead cell dye, and then for CD4 staining without CD8 staining. Naive and memory CD4 T-cell subsets were gated based on characteristic expression patterns of CD45RO and CD27 (or CD28 and CD95 for studies with Sooty mangabeys, as shown in parentheses). (B) Individual subsets of CD4 T cells were analyzed for production of either IL-17 or IFN-γ. (C) The IL-17– and IFN-γ–producing memory subsets were further analyzed for the production of TNF and IL-2.