Depletion of Treg cells enhances the antitumor effects of adoptively infused syngeneic NK cells in tumor-bearing mice. (A) Treg cells and Tconv cells were isolated from BALB/c spleens using immunomagnetic beads selecting for CD4⁺/CD25⁺ Treg cells and CD4⁺/CD25⁻ Tconv cells. T cells were then activated in vitro with anti-CD3 and IL-2 for 4 days. There was a depletion of Treg cells, but not DX5⁺/CD3⁻ NK cells, in mice receiving anti-CD25 antibody or rat IgG isotype antibody. Peripheral blood was harvested 4 days after antibody treatment and was stained for CD25⁺ and CD4⁺ (Treg cells, gated on CD3⁺) or CD3⁻ and DX5⁺ (NK cells). y- and x-axes show log fluorescence (horizontal bars show mean). (B) NK-cell cytotoxicity of RENCA tumor cells in presence of Tconv cells or Treg cells at various T cell–NK cell ratios (1:1, 1:3, and 1:9). A total of 30 μg/mL neutralizing anti–TGF-β antibody was added to 1:1 cocultures of NK cells and Treg cells. BALB/c mice were injected with RENCA (day 0) and treated with anti-CD25 antibody (day 3) and bortezomib (day 4) and syngeneic NK cells (day 5). Tumor progression (C) and survival (D) in mice receiving 3 weekly injections of bortezomib and NK cells; all mice received IL-2. All experiments were performed with 5 or more mice per group and repeated at least once. Results in panel D are pooled from 2 experiments with a total of 8 to 13 animals per group. Values in panels C and D represent tumor-doubling time and median survival days in mice receiving bortezomib and NK cells with anti-CD25 treatment or without anti-CD25 treatment, respectively. P values were calculated by unpaired t test or 2-tailed log-rank test.