Figure 5

Tumor-specific CD4⁺T cell subsets induced by combined therapy. A: Prostate-directed accumulation of HA-specific CD4⁺ T cells. Prostate/prostate-cancer specific CD4 T cells from transgenic mice (6.5 strain) were adoptively transferred to tumor-bearing ProHA x TRAMP mice. After 2 days of in vivo equilibration, mice were treated with GVAX immunotherapy, followed by two doses of anti-CTLA-4 (3 mg/kg) as shown in the top left panel of Figure 5D. Clonotypic (prostate-specific) CD4 were selected by staining for CD4 and the congenic marker Thy1.1. N = 3 animals per group, representative of 2 experiments. *P < 0.01 (GVAX/anti-CTLA-4 vs GVAX alone), **, P < 0.05 (GVAX/anti-CTLA-4 vs GVAX alone). B: CD4 T cell subsets expanded by a combination treatment regimen. HA-specific CD4⁺ T cells were evaluated for secretion of the indicated cytokines by intracellular staining after a brief ex-vivo stimulation. N = 3 animals per group, representative of 2 experiments. *P < 0.05 (GVAX/anti-CTLA-4 vs. GVAX alone). C: Combination treatment expands both specific and endogenous regulatory T cells (Treg). Top panel: experimental design. Bottom Left: HA-specific CD4⁺FoxP3⁺ T cells quantified using intracellular staining of lymphocytes harvested from the indicated organs. Bottom right: Endogenous Treg (experiments performed without adoptive transfer). N = 3 animals per group, representative of 2 experiments.*P < 0.05 (GVAX/anti-CTLA-4 mAb vs GVAX alone). D: Effects of combination treatment on the Effector / Treg ratio. Prostate / prostate-cancer specific CD4⁺ and CD8⁺ effector T cells (IFN-γ positive) were quantified on day 7 following administration of GVAX + anti-CTLA-4 combination therapy, and ratios determined by dividing by numbers of Treg as quantified using FoxP3⁺ intracellular staining. N = 3 animals per group, representative of 2 experiments.