Cytokine secretion by CD4+ T-cell subsets. Peripheral blood mononuclear cells (PBMC) from HLA-DR SE+ rheumatoid arthritis (RA) patients and healthy controls were incubated with 0 or 30 μg/ml citrullinated (cit) fibrinogen or citrullinated aggrecan, stained with mAb directed against (A) CD4, CD28, IFNγ and IL-6 or (E) CD4, CD45RO, IL-6 and IFNγ, and then analysed by flow cytometry. (B) The gating strategy is outlined. FS, forward scatter; SS, side scatter. (C) Fluorescence minus one (FMO) plot showing background staining for intracellular cytokine staining, gated on CD4+ T cells. (D) IL-6 staining of gated CD4- non-T cells. The proportions in (E) of IL-6+CD4+CD45RO+ T cells were 2.2%, 5.3% and 7.1%, and of IL-6+CD4+CD45RO- T cells were 0.8%, 2.1% and 4% in response to incubation with no peptide, citrullinated fibrinogen and citrullinated aggrecan, respectively. Two individual RA patients and one healthy control are shown. Data are representative of two healthy donors and five RA patients.