Figure 3. Antitumor effects of vaccine are mediated by CD8⁺ T cells.

(A) The effect on lifespan of antibody-mediated depletion during the effector phase in vaccinated mice (3 times at 1-week intervals) challenged i.v. with 10⁵ CT26 cells. (B) CD8⁺ T cells were purified from spleens of vaccinated mice, stimulated with γ-irradiated tumor target cells, and then incubated for 48 hours with GFP- or GFP/pFap–transfected CT26 cells. Nuclear apoptosis was assessed by staining with Hoechst 33342 dye as follows: nuclear apoptosis stage 0, no apoptosis; stage 1, large-scale chromatin condensation; stage 2a, chromatin fragmentation; stage 2b, apoptotic bodies. (C and D) Splenocytes from pFap- and empty vector–immunized mice (n = 3) were stimulated for 5 days with pFap-transfected A31 fibroblasts and then subjected to a ⁵¹Cr-release assay. (D) Effector and target cells were coincubated with anti–MHC class I antibodies (mean + SD). (E) FACS analysis of single-cell suspensions of CT26 tumors of vaccinated mice (n = 2) stained with anti-CD3⁺ PerCP-Cy5.5 and anti-CD8⁺ FITC antibodies. One of two experiments is depicted. (F) Representative sections of CT26 tumors of pFap- and empty vector–vaccinated mice stained with anti-CD8⁺ FITC antibodies and DAPI nuclear stain. *Statistically significant compared with vector group, P < 0.01. **Statistically significant compared with empty vector, P < 0.05.