T reg cell inhibition and effector T cell co-stimulation are required for OX86-induced tumor rejection. Rag1-KO C57BL/6 mice were injected i.v. with WT CD8⁺ T cells in addition to a mixture of CD4⁺ T cells comprising 1:10 mixed combinations of CD4⁺ CD25⁺ (Treg) and CD4⁺ CD25⁻ (Teff) cells, respectively, purified from spleens of WT CD45.1 or OX40-KO CD45.2 mice. After 4 wk, mice were injected s.c. with 5 × 10⁵ MCA203 tumor cells and treated with intratumor injection of OX86 when tumors reached 3–4 mm in size. Similarly treated WT and OX40-KO mice were used as controls. Tumor growth is reported in cubic millimeters; each line represents a single tumor. The number of mice rejecting tumor out the total number of treated mice is indicated. When mice were killed, draining lymph nodes were collected and stained to check that the donor T cell mixtures were in the right proportions. A representative plot showing Foxp3 versus OX40 expression (percentages) on gated CD4⁺ T cells is reported for every experimental group. *, P < 0.05.