Fig. 3.

Immunization with KLIpS induces antigen-specific IFN-γ-secreting CD8⁺ T cell population. (A) AAD transgenic and wild-type (WT) mice (n = 6) were immunized twice via footpad with 50 μg KLIpS formulated with PADRE in IFA adjuvant. Seven days after final immunization, 5 × 10⁵ lymph nodes were harvested and cocultured with peptide for 48 hrs in ELISPOT plate. The antigen-specific IFN-γ-secreting spots were developed and analyzed using ELISPOT reader. (Student’s t-test, AAD vs WT, ^**, p < 0.01). The error bar represented the standard deviation (SD) of duplicate experiments. (B) Six AAD transgenic mice were vaccinated with KLIpS, as described in the legend of Fig. 2A. Cross antigen-restimulation was performed to assay the specificity of peptide-induced IFN-γ-secreting cells from peptide-immunized AAD transgenic mice. (Student’s t-testfor KLIpS restimulation: KLIS vs KLIpS immunization; for KLIpS immunization: KLIS vs KLIpS restimulation, ^**, p < 0.01). The error bar represented the standard deviation (SD) of triplicate experiments. (C) Six AAD transgenic mice were vaccinated with KLIpS, as described in the legend of Fig. 2A. Detection of the effector T cell population in 1 × 10⁵ splenocytes of peptide-immunized AAD-transgenic mouse using flow cytometry. The upper-right corners revealed a significant population of stimulated IFN-γ-secreting CD8⁺ T cells among the lymphocytes of KLIpS-immunized AAD transgenic mice. (D) The bar graph shows the percentage of IFN-γ-secreting cells in CD8⁺ T cells (Student’s t-test, KLIS vs KLIpS, ^**, p < 0.01). The error bar represented the standard deviation (SD) of triplicate experiments.