FIG. 5.

ICS of CD8 T cells from C57BL/6 WT or perforin KO mice immunized with the heterologous prime-boost vaccination regimen. C57BL/6 WT or perforin KO mice were immunized with pcDNA3/Adβ-gal or pIgSPCl.9/AdASP-2. Fourteen days after the final immunizing dose, these mice had their splenic cells cultured in the presence of anti-CD28 and brefeldin A, with or without the peptide VNHRFTLV. After 12 h, cells were stained with APC-labeled anti-CD8, fixed, permeabilized, and stained with APC-Cy7-labeled anti-CD3, PE-Cy7-labeled anti-IFN-γ, and Alexa 488-labeled anti-TNF-α. (A and B) Examples of splenic CD3⁺ CD8⁺ cells from immunized C57BL/6 WT or perforin KO mice stained for IFN-γ and TNF-α. (C) Frequency of each cell population. Asterisks indicate that C57BL/6 WT mice immunized with IgSPCl.9/AdASP-2 had a higher frequency of CD3⁺ CD8⁺ T cells expressing either IFN-γ or TNF-α or both cytokines than perforin KO mice immunized with pIgSPCl.9/AdASP-2 (P ≤ 0.01 in all cases). (D) We calculate the frequency of each cell population in relation to the total amount of cells expressing any cytokine. An asterisk indicates that C57BL/6 WT mice immunized with of pIgSPCl.9/AdASP-2 had higher frequencies of CD3⁺ CD8⁺ T cells expressing both IFN-γ and TNF-α than perforin KO mice immunized with pIgSPCl.9/AdASP-2 (P < 0.01 in all cases). A cross indicates that perforin KO mice immunized with IgSPCl.9/AdASP-2 had a higher frequency of CD3⁺ CD8⁺ T cells expressing only IFN-γ than C57BL/6 WT mice immunized with pIgSPCl.9/AdASP-2. The results are presented as medians (bars) and each individual mouse (dots) and are representative of experiments performed twice with similar results.