Enhanced lymphocyte responses in p66Shc^−/− mice. (A) Increased proliferation of B cells from p66Shc^−/− mice. Graph showing percentage (± SD) of proliferating splenocytes from control (+/+, gray line) or p66Shc^−/− (−/−, black line) mice. Proliferation was analyzed by flow cytometry of CFSE-labeled cells after stimulation for 48, 72, and 96 hours with anti–mouse IgM antibodies (30 μg/mL). CFSE fluorescence was analyzed on gated CD22⁺ cells (n ≥ 3). (B, top) Enhanced BCR signaling in p66Shc^−/− B cells. Immunoblot analysis, using a phosphospecific antibody, of Erk1/2 phosphorylation (5 minutes at 37°C) in postnuclear supernatants from splenocytes activated with the indicated concentrations of anti–mouse IgM antibodies (μg/mL). (Bottom) Quantitation by laser densitometry of the relative levels of Erk1/2 phosphorylation in splenocytes activated as described in panel B (top; n = 2, 2 mice/experiment). (C) Amplified response to tetanus toxoid immunization in p66Shc^−/− mice. Serum samples from 6-month-old control (+/+, gray line) or p66Shc^−/− (−/−, black line) mice immunized 4 times with 2 μg TT were collected after each immunization and anti–TT-specific antibodies were measured by ELISA. Representative data of 3 experiments (each carried out on a group of 3 mice) are shown. (D) Enhanced DTH reaction in p66Shc^−/− mice. Control (+/+, gray) or p66Shc^−/− (−/−, black) mice were sensitized with 3% TNCB and challenged 6 days later on one ear (TNCB 1%). Ear swelling was assessed 24 hours later and values are represented as mean of thickness of challenged ear − thickness of unchallenged ear plus or minus SD (n = 6) (*P < .05; **P < .01; ***P < .001).