Fig. 2.

Reduced B and T cell responses in older c-Cbl^−/− mice. (A) FACS plots indicating frequencies of marginal zone B cells (MZB) and follicular B cells (FCB) at 4 (Upper) and 24 weeks (Lower) of age. Analysis was performed on pregated CD19⁺B220⁺ cells. Data are representative of three independent experiments. (B and C) Reduced IgM and IgG antibody responses in older c-Cbl^−/− mice. Five mice per group were injected s.c. with 10 μg of TNP KLH in CFA. At indicated time points after immunization, mice were bled, and the IgM (B) and IgG (C) antibody titers in the serum were determined by ELISA. Error bars indicate the standard errors of the mean. (D) FACS plots indicating frequencies of naÏve (CD62L^highCD44^low/−) and memory (CD44^highCD62L^low/−) CD4⁺ T cells of the spleen at 4 (Upper) and 24 weeks (Lower) of age. Analysis was performed on pregated CD4⁺ cells. Data are representative of five independent experiments. (E) T cell proliferation assays. Sorted naïve CD4 T cells of 4- and 24-week-old c-Cbl^+/+ (filled) and c-Cbl^−/− (open) were CFSE labeled and stimulated with plate bound CD3e (5 μg/mL) and soluble CD28 (2 μg/mL). Three days after stimulation (Left), cells were analyzed by flow cytometry. (F) IL2 secretion assay. Sorted naïve CD4 T cells of 4- and 24-week-old c-Cbl^+/+ and c-Cbl^−/− were stimulated with plate bound CD3e (5 μg/mL) and soluble CD28 (2 μg/mL). Supernatants were collected at 48 hours after stimulation, and IL2 cytokine levels were determined by ELISA. Data are representative of two independent experiments. Shown are the mean values of the triplicate samples. Data are presented as mean ± SEM. *, P values <0.05.