Tumours were harvested at day 17 for analysis of tumour-infiltrating cells. effector cells are in the beginning expanded and can reach the tumour site. Nevertheless, the cytokine and chemokine environment of the tumour is not optimal for the attraction of activated effector cells;6 furthermore, the tumour environment both creates7 and attracts8 suppressive regulatory HEY1 T cells. In these ways, the tumour environment exhibits a pattern of inflammatory resolution. Inflammatory onset is usually associated with expression of pro-inflammatory cytokines such as tumour necrosis factor- (TNF-) and inflammatory mediators such as inducible nitric oxide synthase (iNOS) as part of a general M1 differentiation pattern, whereas inflammatory resolution is associated more with expression of interleukin-10 (IL-10), arginase I and general M2 differentiation.9,10 Unfortunately for the development of T-cell immunotherapies for cancer, a tumour environment of inflammatory resolution does not support effective adaptive immune responses.11 Our laboratory has studied the use of agonistic antibodies to the TNF receptor superfamily member OX40 (CD134) to overcome a lack of adjuvant within the tumour environment antibodies Six to eight week aged C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). OX40-Cre C57BL/6 mice have recently been explained,24,25 and these mice were crossed with B6.129(Cg)-Tg(CAG-Bgeo/GFP)21Lbe/J obtained from The Jackson Laboratory (Bar Harbor, ME) and F1 Cre+ GFP+ mice or control single-positive littermates were used in experiments. These experiments used the MCA205 sarcoma cell collection as previously explained.21 Control RatIg antibody was purchased from Sigma (St Louis, MO) and the rat anti-OX40 antibody (OX86) was produced in the laboratory from hybridomas and affinity-purified over protein G columns. All animal protocols were approved by the Institutions Animal Care and Use Committee. Isolation of tumour-infiltrating cell populations C57BL/6 mice were challenged with 1 106 MCA205 tumour cells subcutaneously ACR 16 hydrochloride in the right flank, which were allowed to establish for 10C14 days. Mice were treated with 250 g ACR 16 hydrochloride OX40 or control antibody intraperitoneally, and 7 days later the tumour was harvested. Isolation of tumour-infiltrating cells was performed as previously explained.21 Briefly, the excised tumour was dissected into 1-mm pieces using crossed scalpels, then digested for 1C2 hr with agitation at room heat in 1 mg/ml collagenase (Invitrogen, Carlsbad, CA), 100 g/ml hyaluronidase (Sigma) and 20 mg/ml DNase (Sigma) in PBS. The resultant preparation was filtered through 100-m nylon mesh and density gradient centrifugation was performed by layering the cell suspension over Ficoll. The resultant buoyant cell layer of tumour-infiltrating cells was washed for use in subsequent experiments. isolation of tumour macrophages Tumour macrophages were isolated from suspensions ACR 16 hydrochloride of tumour-infiltrating cells by plastic adherence or FACS. For isolation by plastic adherence, cells were resuspended in tissue culture media and seeded at a concentration of 1 1 106 cells/cm2 surface area of cell-culture-treated multiwell plates. Cultures were incubated for 30 min at room temperature, then washed three times with media to leave an adherent macrophage population that was 90% CD11b+. For FACS, tumour-infiltrating cells were resuspended in PBS containing 5% fetal bovine serum and stained with CD11b-FITC, Gr1-phycoerythrin and IA-phycoerythrin-Cy5 (all Ebioscience, San Diego, CA). Stained cells were washed, re-filtered over 100-m nylon mesh and the CD11b+ Gr1lo IAhi cell population was sorted on a BD FACSAria to 98% purity. Stimulation of interferon- release by tumour macrophages Macrophages from tumour cell suspensions were isolated by plastic adherence as above or sorted by FACS to 98% pure CD11b+ Gr1lo IAhi cells and seeded to flat-bottomed 96-well ACR 16 hydrochloride plates. Replicate wells were variably treated with 1 ng/ml IL-12 (R&D Systems, Minneapolis, MN) and/or 1 or 10 ng/ml IL-18 (R&D Systems) for 48 hr. Supernatants of treated cells were tested for interferon- (IFN-) secretion by ELISA using matched antibodies from BD Biosciences (San Jose, CA) and compared to a standard curve of recombinant IFN-. For studies, mice bearing MCA205 tumours were treated with 250 g OX40 or control antibody on days.