Phenotypic characterization of the collagen type II–specific type 1 regulatory T cell clones. (A) Graphed data of representative fluorescence-activated cell-sorting (FACS) analysis of the selected clones for the expression of T-cell receptor Vβ8 and CD4. (B) Graph illustrating the results of representative FACS analysis of intracellular cytokine staining of collagen type II–specific type 1 regulatory T cell (Col-Treg) clones following 4 hours of polyclonal stimulation. IFN, Interferon; IL, Interleukin. (C) Graph showing the cytokine secretion profile of three representative Col-Treg clones. The cytokine levels were quantified by enzyme-linked immunosorbent assay in the culture supernatants after 48 hours of polyclonal stimulation. The data are expressed as mean ± SEM. (D) Graph describing the immunosuppressive activity of Col-Treg clones, measured in a cell contact–independent assay by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution, after 3 days of coculture with freshly isolated splenocytes stimulated with anti-CD3 antibody. The data are representative of at least seven clones. Th1, Type 1 T helper cell. (E) Graph of the expression levels of several phenotypic markers following 24 hours of polyclonal stimulation. Values are mean ± SEM of the percentage of positive cells for each marker. The data are representative of three to eighteen clones.