Figure 4: Analysis of cytokine production by intracellular staining (ICS). Results from one representative experiment out of three independent experiments are shown. (a) Splenocytes from mice immunized with IDLV-JRmZ, DNA-JRmZ, IDLV-Empty, and DNA-Empty or from naïve mice were cultured in vitro with Envelope-specific JR-9mer peptide and then stained for IFN 𝛾 , TNF 𝛼 , and IL2. Error bars indicate the standard deviation among four mice of the same group. Data are expressed as percentage of single cytokine-producing T cells by gating the CD8+ T cell population. (b) IDLV-JRmZ vaccination induced high frequency of polyfunctional antigen-specific CD8+ T cells able to simultaneously produce IFN 𝛾 , IL2, and TNF 𝛼 . A representative experiment is shown. The analysis was performed on CD8+ T cells from the mice immunized with IDLV-JRmZ (upper panels) or DNA-JRmZ (lower panels). CD8+/IFN 𝛾 -producing cells were gated and analyzed for the production of both TNFα and IL2. Within the dot plots the percentages of single (IFN 𝛾 ) and double (TNFα and IL2) cytokine-producing CD8+ T cells are indicated. FSC: forward scatter.