FIG. 4.
T-cell responses are similar in WT and GABPβ1L-deficient mice. (A) T-cell proliferation in vitro. Splenic T cells were purified by negative selection and stimulated with plate-bound anti-CD3 alone (1 μg/ml) or in combination with anti-CD28 (1 μg/ml). The proliferation assay was performed in triplicate for each condition with 2 × 105 purified T cells in each well. The cells were activated for 48 h, pulsed with 1 μCi [3H]thymidine, and harvested 12 h later. (B) Detection of antigen-specific CD8 T cells on day 7 p.i. The mice were infected with 5 × 106 actA mutant L. monocytogenes cells expressing OVA, and 7 days later, splenocytes were harvested and cultured in the presence or absence of 200 nM OVA257-264 peptide for 6 h. The cells were stained with anti-CD8 and Thy1.2, followed by intracellular staining of IFN-γ. The percentage of IFN-γ+ cells in the CD8+ population is shown. (Bottom) Background staining in the absence of peptide stimulation. Data are representative of data for at least six mice of each phenotype. (C) Kinetics of CD8 T-cell responses during primary and secondary infection. Antigen-specific T cells were detected as described above (B) on days 7, 14, and 42 p.i. A group of mice of 42 days after primary infection was challenged with 7 × 105 virulent L. monocytogenes cells expressing OVA, and the CD8 T-cell response was determined on day 5 after the secondary infection. Each time point is the average ± standard deviation of data for at least six mice. (D) Detection of antigen-specific CD4 T cells on day 7 p.i. Mice were infected as described above (B), and the splenocytes were stimulated with 5 μM LLO190-201 peptide. The percentage of IFN-γ+ cells in the CD4+ T-cell population is shown. (E) Kinetics of CD4 T-cell responses during primary and secondary infection. Mice were infected as described above (C), and antigen-specific CD4 T cells were detected as described above (D) at various time points.