Characterization of foxp3⁺ Treg in tumor-bearing mice after VLP vaccination.

<p>A). At 12^th day after tumor implantation and after one time VLP vaccination at day 3, mice were sacrificed for immune response analysis. About 10⁶ splenocytes were incubated with the appropriately diluted fluorescent-conjugated antibodies including CD3e, CD4, CD8, and foxp3. Foxp3⁺ cells were analyzed in three T cell subsets: CD4⁺CD8⁻, CD4⁻CD8⁺, and CD4⁻CD8⁻. These were representative data from three independent experiments with at least three mice per group. B). CD4⁺Foxp3⁺ T cells isolated from tumor-bearing splenocytes suppressed Tresp cells proliferation. Co-culturing CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ Tresps (CFSE stained) at different ratios 1∶8, 1∶4, and 1∶2. CFSE labeled Tresps proliferation was analyzed by FACS. Numbers on the CFSE_high peak indicate percent of undivided cells. C). The suppressive activity of CD4⁺CD25⁺ Tregs on CD4⁺CD25⁻ Tresp cell proliferation at different Treg:Tresps ratios (x-axis). Y-axis indicates percent of suppression on Tresp cells proliferation activity.