CD4 T-cell activation by DC vaccination induces CD25⁺ cells capable of suppressing OVA-specific responses. (A) C57Bl/6 mice vaccinated with DCs nucleofected with psigGOL1tail developed large tumors within 2 weeks. If mice were depleted of CD25⁺ cells before vaccination (psigGOL1tail CD25dep), protection was significantly more efficient. P values were calculated using the Student t test. (B) Numbers of CD25⁺Foxp3⁺ cells were equal in mice vaccinated with DCs nucleofected with either construct. The percentages shown are the average of 6 mice per group. (C) OVA-specific suppression of T-cell proliferation by CD4⁺CD25⁺ T cells isolated from mice vaccinated with psigGOL1tail-, but not pGO-nucleofected DCs. CD4⁺CD25⁺ T cells were pooled from 3 mice per group and coincubated with splenocytes from PPD- or OVA-primed mice. PPD- or OVA-induced proliferation is shown as white or black bars, respectively, plus or minus the standard error of mean. (D) DCs nucleofected with either construct had no effect on the protective response against luciferase-expressing B16-F10-luc-G5 melanoma cells in vivo. White bars show the bioluminescence from unimmunized control mice (n = 2) plus or minus the standard error of mean, light gray bars show bioluminescence from mice immunized with tumor-cell lysate (n = 4), dark gray bars show bioluminescence from mice similarly immunized and then vaccinated with DCs nucleofected with pGO (n = 6), and black bars show bioluminescence from mice vaccinated with DCs nucleofected with psigGOL1tail (n = 6).