Figure 2.

CD8⁺ T cells from BALB/c mice immunized with the cathepsin S mutant, VV gp120, generate strong IFNγ responses to peptides containing the IGPGRAFYTT sequence and recognize an IGPGRAFYTT dextramer when compared to mice immunized with the WT gp120 protein. a) BALB/c mice were immunized with either WT gp120 or VV gp120 in CAF09, as described in the Materials and Methods. Splenocytes were isolated 14 days after the final immunization and stimulated in vitro for 6 hrs with (7.5 μg/ml) HIV gp120_MN overlapping 15-mer peptides spanning the length of the gp120_MN protein, and then stained for IFNγ. CD4⁺ T cell responses to peptide pools are shown in Fig S1 b) BALB/c mice were immunized with either WT gp120 or VV gp120 in CAF09 and 14 days later splenocytes were isolated, stimulated in vitro for 6 hrs with (7.5 μg/ml) single peptides 78 (s.p.78), s.p.79, s.p. 80/81 and s.p.TT10 (IGPGRAFYTT) and stained for intracellular cytokines: TNFα, IFNγ and IL-2. c) Mice were immunized with either WT gp120 or the cathepsin S mutant, VV gp120 in CAF09, as described in legends to Figure 1c, and Ag-specific CD8⁺ T cells were detected with D^d-s.p.T10 dextramers. d) Frequency of H-2D^d-s.p.T10 peptide dextramer-binding CD8⁺ T cells isolated from immunized BALB/c mice splenocytes that were directly stained for surface markers, CD3, CD4, CD8 and an H2-D^d s.p.TT10 (IGPGRAFYTT) dextramer. * indicates p < 0.05.