Fig. 3.

Effect of pIL-12 on the frequency of SIV Gag and Env-specific cytokine secreting T cells following EP immunization. Mice were immunized by EP twice, four weeks apart with Gag and Env plasmids and low dose (0.3μg) or high dose (25μg) pIL-12 before a boost with rAd5 vectors expressing Gag and Env four weeks after the second DNA immunization. Splenocytes were harvested at week 6 and week 10, incubated ex vivo for 5h with Gag pooled peptides or the dominant peptides DD13/AL11 (A/C) or Env pool peptides (B/D), anti-CD28 and brefeldin A before being analyzed by flow cytometry for combined production of IFNγ, IL-2 and TNFα. The frequency of multifunctional cytokine secreting Gag-specific (E) or Env-specific (F) CD4+ T cells (%) at week 10 producing IFNγ, IL-2 and TNFα in any combination are shown, bars represent the median, n =4 and data are representative of 2 experiments. *p < 0.05, significantly different from animals immunized by EP or ^#p < 0.05, significantly different from animals immunized by EP+pIL-12 low.