CD4 and CD8 T cells are required for CpG + anti-OX40 + anti-CTLA4 therapeutic effect. (A) Using the 2-tumor site model, mice received either no treatment or CpG + anti-OX40 + anti-CTLA4, with or without CD4 or CD8 depletion. CpG (red arrows) and anti-OX40 + anti-CTLA4 mAb (blue arrows) were administered as previously. CD4 and CD8 depletion (green arrows) was performed by intraperitoneal injections of ascitic fluid containing 0.5 mg of either anti-CD4 mAb (GK1.5 hybridoma) or anti-CD8 mAb (2.43 hybridoma) on days −2, −1, 1, and 5 from start of treatment. Depletion of CD4 and CD8 T cells was confirmed by flow cytometry on peripheral blood. Growth curves represent the total tumor volume (with exception of the right, CpG-injected tumor) expressed as the sum of the surface of the left tumor and all superficial metastatic lymph nodes when present. Numbers indicate the ratio of tumor-free mice at day 100. (B) Alternatively, BALB/c CD8 KO mice bearing 2 tumors received either no treatment or CpG + anti-OX40 + anti-CTLA4 as previously described. Numbers indicate the ratio of tumor-free mice at day 100. (C) Cancer growth was strikingly different between CD4- and CD8-depleted mice after treatment: tumor cells rapidly disseminated to the lymph nodes (LNs) in CD8-depleted mice whereas they remained confined to the primary tumor site (T) in the CD4-depleted mice. Photographs show 3 representative mice from each group.