cDNA (corresponding to 30 ng of RNA), 1.25 ul of 206 Primer/ TaqMan probe mixture, 12.5 ul of 26TaqMan Universal PCR Master Mix and 7.25 ul of DNase/Rnase-free water. All amplifi- cations were done using ABI 7500 Real Time PCR system. Each gene was amplified in triplicate and cDNA concentration differences were normalized to GAPDH. Relative gene expression changes of the target genes in DTA-1-treated group compared to IgG-treated group were calculated by 2-ddCt method (25) using average Ct (threshold cycle) of triplicates from the IgG-treated group as a calibrator. Fold increase or decrease in gene expression was calculated and the values were log10-transformed before plotting. Statistical analysis Differences in long-term tumor-free survival were evaluated by log rank analysis of Kaplan-Meier survival curves (GraphPad Prism 4.0). Statistical differences between groups were determined by analyzing means + standard errors of replicate mice or wells by two-tailed Student’s t test (GraphPad Prism 4.0). Supporting Information Figure S1 DTA-1 induces rejection of B16 melanoma most effectively at day 4. C57BL/6 mice were challenged with 50,000 B16 cells intradermally and treated with DTA-1 or Rat IgG on indicated days. Pooled data from 5 experiments (n = 30250 total mice/group). Mice alive at day 60 without tumor were considered long-term tumor-free survivors. Found at: doi:10.1371/journal.pone.0010436.s001 (0.14 MB TIF) Figure S2 GITR ligation does not decrease Treg expression of Granzyme B, IL10, or TGF-b. For in vitro analysis, purified CD4+foxp3+ cells (from spleens + LN of na¨ıve foxp3GFP mice) were treated with anti-CD3 mAb 1 mg/ml, anti-CD28 mAb 1 mg/ ml, and DTA-1 or Rat IgG 10 mg/ml for 48 hours, followed by RNA extraction and quantitative real-time PCR as per Supple- mental Methods below. For in vivo analysis, RNA was extracted from CD4+foxp3+ cells purified from TDLN of day 10 B16- bearing foxp3GFP mice treated 3 days earlier with DTA-1 or IgG 1 mg i.p. The relative change (on log10 scale) in gene expression (normalized to GAPDH) for DTA-1-treated Tregs compared to IgG-treated Tregs is depicted. Found at: doi:10.1371/journal.pone.0010436.s002 (0.13 MB TIF) Figure S3 No changes seen in absolute number of CD8 T cells/ gram of tumor after DTA-1 treatment. Representative counts from 3 independent experiments showing numbers of T cells per gram of tumor. CD8 T cells are gated on CD45+ CD8+ and Tregs are gated on CD45+CD4+,foxp3+ inside the live gate from tumors, 10 days after B16 inoculation,(6 days post DTA-1 treatment). Found at: doi:10.1371/journal.pone.0010436.s003 (0.19 MB TIF) Figure S4 Abnormal GFP+ Tregs within DTA-1-treated tumors are not undergoing apoptosis. Day 10 B16-matrigel tumors from foxp3GFP mice treated with 1 mg DTA-1 or IgG on day 4 were harvested and processed for TUNEL staining (A) or flow References 1. Nocentini G, Riccardi C (2005) GITR: a multifaceted regulator of immunity belonging to the tumor necrosis factor receptor superfamily. Eur J Immunol 35: 1016–1022. 2. Kohm AP, Williams JS, Miller SD (2004) Cutting edge: ligation of the glucocorticoid-induced TNF receptor enhances autoreactive CD4+ T cell activation and experimental autoimmune encephalomyelitis. J Immunol 172: 4686–4690. 3. Kanamaru F, Youngnak P, Hashiguchi M, Nishioka T, Takahashi T, et al. (2004) Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells. J Immunol 172: 7306–7314. Targeting GITR on Tregs cytometry (B) as per Methods. A) Representative images show lack of TUNEL positive staining of irregular Tregs (arrows) inside DTA1 treated tumors. B) Representative staining of live (DAPI-) tumor-infiltrating Tregs (CD4+GFP+) show no difference in frequency of apoptotic (Annexin V+) cells between DTA-1 and IgG-treated tumors. Gate placement based on fluorescence intensity of stained cells without addition of Annexin V. Found at: doi:10.1371/journal.pone.0010436.s004 (3.93 MB TIF) Figure S5 Schema for reconstitution of RAG12/2 mice with GITR2/2 or GITR+/+ effector (Teff) or regulatory (Treg) T cells. Found at: doi:10.1371/journal.pone.0010436.s005 (0.18 MB TIF) Figure S6 In vivo GITR ligation does not alter surface expression of CD103, CD62L, or CCR7 on Tregs within tumor-draining lymph nodes. B16-bearing C57BL/6 mice were treated with DTA-1 or IgG 1 mg on day 4 and tumor-draining lymph nodes harvested 48 hours later. Isolated lymphocytes were stained for FACS. Expression of indicated molecules on gated live CD4+foxp3+ Tregs from representative mice are depicted. Similar findings were observed 72 hours after DTA-1 or IgG treatment (data not shown). Found at: doi:10.1371/journal.pone.0010436.s006 (0.30 MB TIF) Figure S7 GITR ligation blocks TGF-b-mediated in vitro peripheral conversion to induced Tregs. 56104 CD4+foxp3- cells (FACS-sorted from naive foxp3GFP splenocytes) were cultured for 5 days at 37uC with 1.56105 irradiated T-cell depleted splenocytes, 0.1 mg/ml anti-CD3 mAb, 1 mg/ml anti-CD28 mAb, and indicated concentrations of IgG, DTA-1, or OX86 (agonist anti-OX40 mAb). 40 U IL-2 and 5 ng/ml TGF-b1 was added to each well after 48 hours in culture. After a total of 5 days incubation, cells were harvested, stained with anti-CD4 and DAPI and analyzed by FACS. The % foxp3+GFP+ from gated CD4+DAPI- are shown. Similar findings were seen using anti- CD3 mAb at 0.01 or 1 mg/ml (data not shown). Data are from 1 of 3 representative experiments. Found at: doi:10.1371/journal.pone.0010436.s007 (0.43 MB TIF) Acknowledgments The authors wish to thank Hong Zhong and Andre Burey for their management of mouse colonies. Dr. Francesca Avogadri, Dr. Karim Lakehal for assistance with sample preparation and T cell transfers, along with input regarding experimental design. Dr. Li-Fan Lu, and Dr. Ashutosh Chaudhry for input regarding experimental design. MSKCC Molecular Cytology Core Facility for assistance with confocal microscopy. Dr. Stephanie Terzulli, Dr. Alan B Frey and Dr. Li-Fan Lu for their comments and careful reading and editing of this manuscript. Author Contributions Conceived and designed the experiments: ADC DAS TM ANH JDW. Performed the experiments: ADC DAS CL YL DHC SCK AD GAR FD. Analyzed the data: ADC DAS CL SCK MP TM ANH. Contributed reagents/materials/analysis tools: TM. Wrote the paper: ADC DAS. 4. Ronchetti S, Zollo O, Bruscoli S, Agostini M, Bianchini R, et al. (2004) GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Eur J Immunol 34: 613–622. 5. Tone M, Tone Y, Adams E, Yates SF, Frewin MR, et al. (2003) Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc Natl Acad Sci U S A 100: 15059–15064. 6. Stephens GL, McHugh RS, Whitters MJ, Young DA, Luxenberg D, et al. (2004) Engagement of glucocorticoid-induced TNFR family-related receptor on effector T cells by its ligand mediates resistance to suppression by CD4+CD25+ T cells. J Immunol 173: 5008–5020. PLoS ONE | www.plosone.org 11 May 2010 | Volume 5 | Issue 5 | e10436