Figure 4. Effects of sPD-1 and sTim-3 on CD4⁺ and CD8⁺ T cells from mice immunized with rAd5-SIV vaccine in producing IFN-γ and proliferation capability upon stimulation with SIV antigen peptides. (A) The percentage of antigen specific IFN-γ⁺ CD4⁺ T cells in total CD4⁺ T cells. (B) The percentage of antigen specific IFN-γ⁺ CD8⁺ T cells in total CD8⁺ T cells. (C) The percentage of antigen specific proliferating CD4⁺ T cells. (D) The percentage of antigen specific proliferating CD8⁺ T cells. C57BL/6 mice were immunized with either rAd5-SIV alone or rAd5-SIV co-administered with rAd5-sPD1 or rAd5-sTim3, or rAd5-SIV co-administered with both rAd5-sPD1 and rAd5-sTim3. At 2 wk after immunization, splenocytes were harvested and cultured with SIV Gag peptides. A flow cytometry assay based on intracellular cytokine staining was used to quantify Gag-specific IFN-γ secreting CD4⁺ and CD8⁺ T cells. The percentage of proliferating cells was determined by the ratio of the CFSE-low cells to the total CD4⁺ T cells or total CD8⁺ T cells respectively. The data are the representation of two independent experiments.