Figure 6.

Vaccine-Primed Anti-FSHR CD8⁺ T Cells Are Necessary for the Effect of the FSHR SynCon Vaccine and Able to Delay FSHR⁺ Tumor Progression

(A) Schematic of tumor challenge depletion experiment: we vaccinated mice and challenged with ID8-Defb29/Vegf-a-Fshr 1 week after the last immunization. At 1 day prior to the tumor challenge and twice weekly thereafter, we administered either anti-mouse CD8 or rat polyclonal IgG. (B) Survival plot of the FSHR SynCon vaccine or pVAX empty vector with our without CD8 depletion (single experiment with n = 5 mice per group). (C) Interferon-γ ELISPOT of splenocytes from mice treated with murine FSHR consensus vaccine pulsed with murine FSHR peptides (one experiment with n = 4 mice). (D) Percentage of CD8⁺ T cells producing IFNγ upon stimulation with STYRLKKL peptides at different concentrations (representative of 2 independent experiments). The T cells of this experiment were expanded with KKLRARSTYRLKKLP peptide except for those in the last column, which were expanded with IFTKNFRRDFFVLMS. (E) Histogram and representative flow cytometry plots of H2-K(b)-STYRLKKL tetramer CD8⁺ T cells in peripheral blood of mice vaccinated with FSHR consensus vaccine, native vaccine, or pVAX 90 days after first vaccination. (F) Cytotoxicity of T cells derived from murine FSHR SynCon vaccine expanded with KKLRARSTYRLKKLP peptide (5 ng/mL) 4 weeks after starting peptide stimulation of ID8-Defb29/Vegf-a or ID8-Defb29/Vegf-a-Fshr tumor cells, measured by 7-AAD/Annexin V flow cytometric staining after co-culture of 5 hr (representative of 2 independent experiments). (G) Survival plot of mice challenged with 2 million ID8-Defb29/Vegf-a-Fshr cells and treated a day later with 2 million FSHR_STYRLKKL-specific T cells expanded ex vivo (n = 10 mice), anti-mouse CD3/CD28 bead-expanded T cells (n = 5 mice), or vehicle (PBS; n = 5 mice) (log rank, one-way ANOVA, and unpaired t test). Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001.