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, TNF 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, IL2, and TNF. 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-producing cells were gated and analyzed for the production of both TNFα and IL2. Within the dot plots the percentages of single (IFN) and double (TNFα and IL2) cytokine-producing CD8+ T cells are indicated. FSC: forward scatter.