B-CD8+ T Cell Interactions in the Anti-Idiotypic Response against a Self-Antibody
P3 mAb was able to recognize and activate in vitro B-2 cells. (a) Splenic B-2 cells isolated from naïve BALB/c mice were incubated with biotinylated P3 or control IgM mAbs, and the binding was evaluated by flow cytometry using a PECy5-conjugated streptavidin. Contour plots (left) show the results obtained for a representative mouse; columns (right) represent means ± SD of the values obtained for three mice. (b-c) Splenic B-2 cells obtained from naïve BALB/c mice were incubated three days with 100 μg/mL of P3 mAb or IgM isotype control. Expression of the activation markers CD25, CD69, and CD86 was measured by flow cytometry (b), and cytokine production (IFN-γ, IL-4, and IL-10) was measured by flow cytometry after an intracellular staining protocol (c). (d) The IgG subclasses of the response against P3 mAb were determined in mice immunized subcutaneously with four doses of P3 mAb in PBS every two weeks. Sera samples were taken after the fourth dose (day 49). Sera were diluted to 1 : 100, and the reactivity against murine P3 mAb was assessed by ELISA using biotin-conjugated rat anti-mouse IgG1, IgG2a, IgG2b, or IgG3, followed by alkaline phosphatase-conjugated streptavidin. Means ± SD of the values obtained in triplicate for each individual mouse are graphed, , Mann–Whitney U test (a–c) or Games-Howell test (d), both one-tailed. Different letters indicate statistical difference among the groups (d).