In experimental models

of immune activation, Tem cells constitutively express CD40L at levels sufficient to induce DC activation in an antigen-independent manner 17. The CD40/CD40L axis is crucial for DC maturation and the subsequent T-cell priming. However in the tumor microenvironment this costimulatory pathway is often dampened, thus impairing the generation of an efficient anti-tumor immune response 18, 19. In this study we have investigated the mechanisms by which OX86 modulates Treg- and Teff-cell functions and their reciprocal interactions with DCs at the tumor site. We propose a model of the tumor microenvironment in which, after OX86 treatment, DCs receive a lower IL-10-mediated inhibition by Treg Everolimus in vivo cells on the one hand, and a stronger stimulation from Tem cells, via the CD40/CD40L axis, on the other. In this favorable condition, DCs acquire a stronger migratory ability toward the draining LNs (dLNs), thus inducing a specific anti-tumor immune response. Intratumoral OX40 triggering promotes tumor rejection modulating both Treg- and Teff-cell functions 3, through unknown mechanisms. Here, we separately analyzed the consequences of OX40 triggering on Treg and Teff cells. Treg cells infiltrating the transplantable CT26 colon

carcinoma expressed OX40 at higher levels than Treg cells in dLNs (Fig. 1A). We evaluated IL-10 secretion as part of the Treg-cell-suppressive activity directly ex vivo. Low levels of IL-10 were produced by Treg cells in dLNs (Fig. 1B and C), whereas about 40% of tumor-infiltrating Treg cells spontaneously produced IL-10 (Fig. 1D and E). beta-catenin mutation Twenty-four hours after OX86 treatment, IL-10 secretion by tumor-infiltrating Treg cells was significantly decreased (Fig. 1D and E). Similar

results were obtained also in mice bearing TSA mammary carcinoma (Supporting Information Fig. 1). Some authors have reported tumor-infiltrating CD11b+CD11c+ cells expressing OX40 20, while others did not detect OX40 expression on CD11b+ cells, even if OX86 systemic administration could indirectly reduce their frequency in tumors 21. Tumor-infiltrating macrophages (CD45+CD11b+F4/80+), Y27632 representing the vast majority of immune infiltration in our tumor model, neither expressed OX40 nor was their IL-10 secretion affected by OX40 stimulation (data not shown). The decreased IL-10 production by Treg cells upon OX40 engagement was confirmed with a different experimental approach. BM chimeras were generated such as to carry an IL-10-GFP reporter transgene 22 in the hemopoietic lineage. IL-10-GFP expression, evaluated in tumor-infiltrating CD4+CD25high Treg cells, was significantly reduced after intratumoral OX86 injection (Fig. 1F and G). Unfortunately, we could not finely locate IL-10-GFP expression into the Foxp3+-gated Treg-cell subset, since the fixation step required for Foxp3 detection led to GFP loss (data not shown).