Figure 4. Effect of immunization with PA on PA-reactive T cells in vivo.

(A) B6 mice were injected with vehicle (Veh) or PA (20 μg i.p.), and liver was harvested 24 h later. Isolated liver MNCs were stained with CD1d tetramers loaded with PBS or PA. Numbers on plots indicate % TCRβ⁺tetramer⁺ cells of live non-B lymphocytes. Dotplots shown are representative of four independent experiments, each using 2 to 3 mice per group. Percent positive and absolute numbers of PA/CD1d-tet⁺ cells from these animals are expressed as the mean ± SE in a bar diagram (*p <0.003; n = 9 mice/group, pooled from three experiments). (B) Liver MNCs from these animals were stained with CD1d tetramers loaded with PBS57 and PI, and analyzed on live non-B lymphocyte gate. Numbers on plots indicate % TCRβ⁺tetramer⁺ cells of live non-B lymphocytes. Data shown are representative of three experiments. (C) PA/CD1d-tet⁺ T cells from these animals were analyzed for CD69 expression (*p <0.0001; n = 9 mice/group; mean ± SE). (D-F) Liver MNCs isolated 24 h after a PA or vehicle injection were cultured for 5–6 h with 500 ng/ml ionomycin and 10 ng/ml PMA in presence of Golgi-plug (1 μl/ml), and cells analyzed for intracellular cytokines. Results of IFNγ production on gated TCRβ⁺ PA/CD1d-tet⁺ cells (D, E) or gated iNKT cells (F) are shown. Numbers on histograms represent % positive cells or MFI, as indicated. Data in (E) are presented as the mean ± SE % positive and MFI of IFNγ on gated PA/CD1d-tet⁺ cells (*p <0.002; n = 6 mice/group, pooled from three experiments). Data shown are representative of three independent experiments. (G) B6 mice were injected with vehicle (Veh) or PA (20 μg i.p.), and bled at the indicated timepoints. Their serum samples assayed by ELISA for cytokines (*p <0.001; n = 6–8 mice/timepoint, pooled from three separate experiments; mean ± SE).