Antigen-induced T-cell responses measured in PLN slices are decreased by PTX. (A) Average Ca²⁺ responses of Marylin TCR-tg CD4⁺ T cells incubated with suB or PTX and stimulated with DBY peptide. Fura-2-loaded T cells were incubated for 10 minutes with 100 ng/mL suB (black line) or PTX (gray line), washed, and overlaid on PLN slices. Time-lapse imaging was started 2 hours after suB or PTX treatment. After several minutes of imaging, the preparation was perfused with a solution containing 100 nM DBY peptide. Ca²⁺-signals of responding T cells were synchronized at the rising phases of the response. (B) Percentage of Ca²⁺-responding CD4⁺ T cells induced by DBY peptide (100 nM). Mean plus or minus SD of 3 independent experiments in which more than 50 cells were analyzed per experiment. *P < .05. (C) CD69 expression measured by flow cytometry on T cells activated within PLN slices. Marylin CD4⁺ TCR-tg T cells were incubated with 100 ng/mL of suB or PTX, labeled with CMFDA and overlaid on PLN slices. Two hours after DBY peptide treatment, slices were mechanically dissociated and recovered cells stained with anti–CD69-PE and anti–CD45.1-PE-Cy5 Abs. Results in the left panel are representative of 3 independent experiments and show percentage and MFI of CD69⁺ cells. The right panel shows combined MFI of CD69 expression after normalization to the value of suB-treated T cells. *P < .05.