Tumor growth in mice depleted for CD4, CD8, or NK cells. Neu-tg mice received an intraperitoneal injection of anti-CD4, anti-CD8, and anti-GM1 three days before transplantation of MMC-Rlx cells (pool) and then every fourth day. Control mice were injected with isotope control antibody. All mice received Dox. (A) Tumor size at day 14 after injection (n = 5 per group). Significant differences of P < .001 are indicated by an asterisk. (B) Quantification of immune cells in MMC and MMC-Rlx tumors 3 weeks after tumor cell transplantation. All mice received Dox, starting at day 2 after transplantation. Tumor-infiltrating lymphocytes were extracted and analyzed by flow cytometry for the frequency of CD4+ and CD8+ cells in TILs. Differences between groups were not significant. (C) Rechallenge study. Mice received a subcutaneous injection of 105 MMC-Rlx29 cells into the left inguinal region (○) or a PBS injection (■). Both groups were treated with Dox as described in Figure 2F. Two weeks after injection of PBS or MMC-Rlx29 cells, mice received a subcutaneous injection of 5 × 105 MMCs into the right inguinal region. Shown is the volume of challenge tumors at different days after MMC cell implantation. n = 5 per group. Error bars represent SD. (D) Combination of Rlx expression and Treg depletion by low-dose cyclophosphamide. Mice with tumors derived from MMCs or MMCs expressing Rlx (MMC-Rlx) received drinking water with or without Dox, starting day 2 after implantation. At day 12 after tumor transplantation, mice were injected intraperitoneally with 2 mg cyclophosphamide (CY) or PBS. Kaplan-Meyer survival data are shown. End point was the day when tumors reached a size of 500 mm3 (n = 7 per group).
