T-cell response after immunization with glucose-6-phosphate isomerase or arthritogenic peptides. DBA/1 mice were immunized with 400 μg glucose-6-phosphate-isomerase (G6PI), 50 μg G6PI85–99, 50 μg G6PI325–339 or 50 μg G6PI469–483 in complete Freund's adjuvant. Mice were sacrificed on day 12 after immunization and single-cell suspensions from draining lymph nodes were prepared. Cells were stimulated with same antigen as used for immunization (20 μg/ml G6PI or 5 μg/ml peptide). (a) Antigen-specific CD4+ cells were identified by CD154 expression as described in Materials and methods. Numbers above the gates indicate the percentage of CD154+ cells. (b) Total numbers of antigen-specific CD4+ cells were calculated by multiplication of the total number of lymph node cells with the frequency of CD4+CD154+ cells. (c) Intracellular staining for TNFα, IL-17, RANKL and IFNγ. Dot plots show the expression of these cytokines in antigen-specific T cells (gated on CD4+CD154+ cells). Numbers in quadrants indicate the percentage of cells producing the respective cytokines within the antigen-specific T-cell compartment. Data depicted in (a) and (c) are from concatenated data files from five individual mice per group and represent all mice in the respective group. This experiment was performed at least two times. FSC, forward scatter; ns, nonsignificant; P > 0.05 between G6PI-immunized vs. peptide-immunized mice.