in our RAG12/2 mice and have been reported recently to have augmented activation with GITR co-stimulation.[17,45]. Regard- less, these data suggest that GITR must be present on both Treg and Teff populations for the full anti-tumor effects of DTA-1. In conclusion, a single dose of agonist anti-GITR mAb can induce T-cell-mediated rejection of an aggressive, poorly immu- nogenic tumor. Additionally, our data supports the idea that DTA- 1 modulates both Teffs and Tregs during therapy. GITR ligation on Tregs appears to cause impaired accumulation within the tumor through Treg lineage instability and a block of trafficking. This leads to greater intra-tumor Teff:Treg ratios and more potent Teff activity. Furthermore, those Tregs which do traffic to the tumor are likely limited in their suppressive capacity as denoted by loss of continued nuclear foxp3 protein expression. Importantly, the preservation of global Treg function and limitation of this skewed ratio to the tumor may make widespread loss of tolerance and autoimmune consequences with in vivo GITR ligation less likely. These data provide further support for the development of this strategy as a therapeutic approach to human cancer. Materials and Methods Ethics Statement Mice were maintained according to NIH Animal Care guidelines, under a protocol 96-04-017 approved by the MSKCC Institutional Animal Care Committee. Mice C57BL/6 Thy1.2+ and Thy1.1+ mice were obtained from Jackson Laboratory (Bar Harbor, ME). Thy1.1+ pmel-1 T-cell receptor transgenic mice have been reported.[46] foxp3GFP knock- in mice were a gift from Dr. A. Rudensky (MSKCC, NY, NY).[20] GITR2/2 and GITR+/+ littermates (Sv129 x C57BL/6 back- ground)[47] were a gift from Dr. P.P. Pandolfi (MSKCC, NY, NY) and were backcrossed .10 generations onto C57BL/6 back- ground using a speed congenic system[48]. Cell lines and tumor challenge B16F10/LM3 (hereafter called B16) is derived from the B16F10 line provided by I. Fidler (M.D. Anderson Cancer Center, Houston, TX). EL4, (American Type Culture Collection, ATCC Number: TIB-39, Manassas, VA), C57BL/6 lymphoma cell line was used as an antigen-presenting cell in intracellular cytokine assays. Cell lines were cultured as described.[49] For tumor-free survival experiments, 50,000 B16 cells were injected intradermally in serum-free RPMI media, and tumor diameters were measured with calipers every 2–3 days. For experiments requiring recovery of tumor-infiltrating lymphocytes (TIL), 500,000 B16 cells in growth factor-reduced, phenol red-free Matrigel (BD Biosciences) were injected subcutaneously. Lymphocyte isolation Spleens, tumor-draining inguinal lymph nodes, and tumors were homogenized through 0.22 mm strainers to produce single cell suspensions. RBCs were lysed from spleens using an ammonium chloride lysis buffer. TILs were isolated from tumor suspensions by density gradient centrifugation using Percoll (GE Healthcare, Piscataway, NJ). Briefly, cell pellets were suspended in 80% Percoll, overlayed with 40% Percoll, and centrifuged at 20006g for 30 minutes. Cells at the interface were collected, washed, and used for FACS or functional assays. Intracellular cytokine assays for expression of IFN-c and CD107a following peptide restimulation were performed as reported.[16] Targeting GITR on Tregs Isolation of CD8+, CD4+foxp3+, and CD4+foxp3- cells from foxp3GFP mice was performed on a Cytomation MoFlo cell sorter. Isolation of CD8+, CD4+CD25+, and CD4+CD25- cells from other mouse strains was performed using MACS microbead separation kits (Miltenyi Biotec Inc., Auburn, CA). pmel-1 CD8+ cells were labeled with CFSE 2.5 mM prior to adoptive transfer. Transferred cells were injected intravenously by tail vein in 200 ml sterile PBS. For some adoptive transfer experiments, recipients were pre-treated with cyclophosphamide (Sigma-Aldrich) 250 mg/ kg in 500 ml sterile PBS intraperitoneally one day prior to transfer. Antibodies and FACS analysis The DTA-1 hybridoma, from S. Sakaguchi (Kyoto University, Kyoto, Japan), and the OX86 hybridoma, from A. Weinberg (Earle Chiles Research Institute, Portland, OR), were used to produce agonist anti-GITR and anti-OX40 mAb, respectively, by the MSKCC Monoclonal Antibody Core Facility. One mg affinity-purified DTA-1 in 500 mL PBS was injected intraperito- neally. Purified Rat IgG (Sigma-Aldrich) was used as a control. Foxp3 staining was performed using the Mouse Regulatory T-Cell Staining Kit (eBioscience, San Diego, CA). Other flow cytometry antibodies were from BD Biosciences (San Jose, CA). hgp10025-33- Db-tetramer, containing the Db epitope KVPRNQDWL, has been described.[16] For fixed/permeablized samples, LIVE/DEADH Fixable Dead Cell Stain Kit (Invitrogen, Carsbad, CA) was used to identify dead cells; for other samples DAPI was used. Samples were run on a BD 4-color FACSCalibur or 12-color LSRII cytometer, and were analyzed using FlowJo (Treestar, San Carlos, CA). Immunofluorescence B16-matrigel tumor samples were harvested on day 10 and fresh frozen tissue was embedded in OCT. Sequential 5 mm sections were collected and stained with anti-CD31 biotin (BD Pharmin- gen), anti-CD8 Alexa fluor 657 (Caltag/Invitrogen), anti-foxp3 APC (ebiosicence), anti-Caspase 3 (BD Pharmingen) and DAPI for nuclear stain. TUNEL staining (TdT and biotin-dUTP from Roche Diagnostics Corp.) was performed after proteinase K digestion (Sigma) and detected using Alexa-633 Avidin (Molecular Probes). Images were acquired with a Leica upright confocal microscope using 20X objective at 204862048 resolution. Acquisition was performed using IgG-treated LN sections to set PMT detector settings at non-saturating conditions, with back- ground levels set using isotype control or secondary antibody alone. Detector voltages were then maintained for each staining condition across all sections. Treg suppression assay 105 CD8+ Teff were labeled with 1.25 mM CFSE and cultured with CD4+foxp3+ cells (FACS-sorted from foxp3GFP mice) or CD4+CD25+ cells (MACS-selected from C57BL/6 mice) at indicated ratios, along with 105 T-cell-depleted, irradiated (444 cGy) splenocytes and 1 mg/ml anti-CD3 mAb. After 4 days, cells were harvested, stained with anti-CD8 and DAPI and analyzed by FACS. Quantitative real time PCR Total RNA was extracted from FACS-sorted Tregs using RNeasy Micro Kit (Qiagen, CA) and cDNA synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA) according to manufacturer’s instructions. All primers and probes were from TaqManH Gene Expression Assays (Applied Biosystems). Real-time PCR reactions were prepared with 4 ul PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10436