FIG. 2.

Induction of gp120-specific CD8⁺ T-cell responses by gp120-NPs. Mice were intranasally immunized once with the indicated antigens. Spleen lymphocytes were isolated on day 10 after immunization. (A) Antigen-specific CD8⁺ T cells were detected by H-2D^d/p18 pentamer staining. Data are expressed as the percentages of the gated CD8⁺ T cells that bound to the pentamer, as measured by flow cytometry. All data represent the mean ± SEM of the results for four mice per group. Statistical analysis was carried out in comparison with the results for the gp120 + CTB group. *; P < 0.05; **; P < 0.001. (B) Spleen lymphocytes were evaluated by ELISPOT for IFN-γ production after stimulation with no peptide (−), the p18 peptide (5 μg/ml), or recombinant gp120 (rgp120; 5 μg/ml). Data are expressed as the numbers of antigen-specific spots per million cells. All data represent the mean ± SEM of the results for four mice per group. Statistical analysis was carried out in comparison with the results for the gp120 + CTB group. *, P < 0.05; **, P < 0.001. (C) CD8⁺ T cells were evaluated by intracellular cytokine staining for the production of IL-2, IFN-γ, and TNF-α after stimulation with the p18 peptide. Data are expressed as the percentages of cytokine-positive CD8⁺ cells. (D) Spleen lymphocytes were cultured for 4 days in the presence of the p18 peptide (10 μg/ml). The cells were harvested and used as effector cells to assess P815 target cell lysis by measuring LDH release after overnight incubation with medium alone or the p18 peptide. Data are expressed as the levels of peptide-specific lysis, calculated by subtracting the percent specific lysis of the control target cells from the percent specific lysis of the peptide-pulsed target cells at the indicated effector-to-target (E:T) cell ratio.