Vaccine-Induced Anti-Neuroblastoma Immunity Early after HSCT Arises in the Presence of Foxp3⁺CD25⁺CD4⁺ T Cells.

Effective anti-neuroblastoma immunity can be induced in mice as early as 1 week after syngeneic HSCT by vaccination with neuroblastoma cells transfected to express CD80, CD86, CD54, and CD137L. However, effective vaccination at this early time point also requires adoptive transfer of T cells (2×10⁷ splenocytes). Previous results in non-transplanted mice showed that T-reg depletion/blockade with anti-CD25 mAb (clone PC61) increased tumor vaccine efficacy. To determine if PC61 treatment might be effective early after HSCT, we first determined if CD4⁺CD25⁺Foxp3⁺ T-reg cells were present, derived either from host (i.e., they survived lethal TBI) or from the transferred spleen cells. Even though transferred splenocytes accelerated T cell reconstitution, HSCT recipient mice remained severely lymphopenic until 3 weeks after transplant. Furthermore, the percentage of Foxp3⁺ cells in the splenic CD4 fraction was significantly increased in transplanted mice, being as much as 3 times higher than that in non-transplanted mice (35% vs. 12%). During this time, the majority of CD25⁺Foxp3⁺CD4⁺ cells were derived from the adoptively-transferred T cells. Despite the presence of CD25⁺Foxp3⁺CD4⁺ T cells, protective anti-tumor immunity could still be efficiently induced. To examine whether depletion/inhibition of CD25⁺ T-reg cells could further enhance vaccine-induced immunity in transplanted mice, two strategies were tested: (a) physical depletion of CD25⁺ regulatory T cells from the adoptively-transferred T cells, and (b) anti-CD25 mAb treatment of the transplanted mice 4 days after HSCT to target both transferred and residual endogenous CD25⁺ cells. Immunomagnetic depletion eliminated more then 95% of the CD25⁺ T cells in the transferred population, but only 70% of Foxp3⁺CD4⁺ T cells. Adoptive transfer of CD25-depleted splenocytes at the time of HSCT resulted in modest increased tumor vaccine efficacy as compared to mice given non-depleted splenocytes. Two weeks after HSCT, both groups of mice contained similar percentages of Foxp3⁺CD4⁺ T cells in the blood and lymph nodes, but a lower frequency of Foxp3⁺CD4⁺ T cells was detected in the spleens of mice given CD25-depleted splenocytes (35% vs. 43%, p<0.01). Our second strategy, treatment of HSCT recipients with anti-CD25 mAb prior to tumor vaccination, negatively influenced the vaccine-induced anti-tumor response and resulted in decreased survival. This suggested that the antibody treatment inhibited effector cell function in the early post-transplant setting. IFN-γ ELISpot analysis demonstrated that vaccinated transplant recipients given anti-CD25 mAb had significantly decreased numbers of tumor-specific CD4 T cells in spleen. Collectively, these results suggest that anti-neuroblastoma immunity could be induced in the present of CD25⁺Foxp3⁺CD4⁺ T-reg cells early after HSCT; strategies that targeted at CD25 did not significantly increase the efficacy of the tumor vaccine.