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Supplementary Materials and Methods Transfection and expression of plasmids ProIL-33 and mtrIL-33 construct expression was confirmed by western blot and immunofluorescence microscopy. Human Rhabdomyosarcoma (RD) cell lines were cultured, transfected and harvested as described previously (1). Briefly, RD cells were cultured in 6-well plates and transfected with the constructs (pVAX as control) using LipofectamineTM2000 (Invitrogen) following the manufacturer’s protocol. Forty-eight hours later cells were lysed using modified RIPA cell lysis buffer and cell lysate was collected. Western blot analysis was performed with an anti-IL33 monoclonal antibody (R&D systems) and visualized with horseradish peroxidase (HRP)-conjugated anti-rat IgG (Cell Signaling) using an ECL western blot analysis systems (GE Amersham). In addition, supernatants were also collected at 48 hours after transfection and cytokine secretion was examined by mouse/rat IL-33 Quantikine ELISA kit (R&D Systems) according to manufacturer’s protocol. An indirect immunofluorescence microscopy assay was also utilized to confirm expression of both IL-33 isoforms as described previously (2). Briefly, RD cells were plated on two-well chamber slides (BD Biosciences) and grown to 70% confluence overnight in a 37 incubator with 5% CO2. The cells were transfected with 1ug of IL-33 constructs and the control plasmid pVAX (1 ug/well) using TurboFectinTM8.0 Transfection Reagent (OriGene) according to the manufacturer’s instructions. Forty-eight hours later the cells were fixed on slides using ice cold methanol for 10 min. The cells were stained with anti-IL-33 mouse monoclonal antibody (R&D Systems, Minneapolis, MN) and subsequently incubated with Alexa 555-conjugated anti-rat secondary antibody (Cell Signaling). Slides were mounted using Fluoromount G with Dapi (Southern Biotechnology). Images were analyzed by florescence microscopy (Leica DM4000B, Leica Mcirosystems Inc, USA) and quantification was conducted using SPOT Advanced software program (SPOTTM Diagnostic Instruments, Inc). Intracellular cytokine stain for Flow Cytometry Splenocytes were added to a 96-well plate (1x105/well) and were stimulated with pooled HPV16 E6/E7 pooled peptide for 5-6 hours at 37C/5% CO2 in the presence of Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin) (eBioscience) according to the manufacturers instructions. The Cell Stimulation Cocktail (plus protein transport inhibitors) (phorbol 12myristate 13-acetate (PMA), ionomycin, brefeldin A and monensin) (eBioscience) was used as a positive control and R10 media as negative control. In cultures being used to measure degranulation, anti-CD107a (FITC; clone 1D4B; Biolegend) was added at this time to enhance staining. All cell were then stained for surface and intracellular proteins as described previously (3). Briefly, the cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) before surface staining with flourochrome-conjugated antibodies. Cells were washed with FACS buffer, fixed and permeabilized using the BD Cytofix/Ctyoperm TM (BD, San Diego, CA, USA) according to the manufacturer’s protocol followed by intracellular staining. The following antibodies were used for surface staining: LIVE/DEAD Fixable Violet Dead Cell stain kit (Invitrogen), CD19 (V450; clone 1D3; BD Biosciences) CD4 (V500; clone RM4-5; BD Biosciences), CD8 (PE-TexasRed; clone 53-6.7; Abcam), CD44 (A700; clone IM7; Biolegend); KLRG1 (FITC; clone 2F1; eBioscience); PD-1 (PeCy7; clone RMP1-30; Biolegend). Major histocompatibility complex class I peptide tetramer to LCMV-GP33 was used as described previously (4,5). For intracellular staining the following antibodies were used: IFN-γ (APC; clone XMG1.2; Biolegend), TNF-α (PE; clone MP6-XT22; ebioscience), CD3 (PerCP/Cy5.5; clone 145-2C11; Biolegend). All data was collected using a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR) and SPICE v5.2 (free available from http://exon.niaid.nih.gov/spice/). Boolean gating was performed using FlowJo software to examine the polyfunctionality of the T cells from vaccinated animals. Dead cells were removed by gating on a LIVE/DEAD fixable violet dead cell stain kit (Invitrogen) versus forward scatter (FSC-A) Ag-specific antibody determination The measurement of IgG antibodies specific for viral genes E6 and E7 was performed by ELISA (enzyme linked immunosorbent assay) in both immunized and controlled mice. The plates were coated with 1ug/ml of each protein (ProteinX Lab) and incubated overnight at 4 degrees. After washing, plates were blocked with 10% fetal bovine serum (FBS) in 1x phosphate-buffered saline (PBS) for 1 hour at room temperature. Plates were then washed again and serum was added at a 1:25 dilution in 1% FBS + PBS + 0.05% Tween-20 and incubated at room temperature for 1 hour. After another wash, goat anti-mouse IgG HRP (Santa Cruz) at a 1:5000 dilution was added to each well and incubated for 1 hour at room temperature. Following a final wash, the reaction was developed with the substrate 3,3’,5,5’-tetramethylbenzidine (SigmaAldrich) and stopped with 100ul of 2N sulfuric acid/well. Plates were read at 450nm on Glomax Multi-Detection System (Promega). All serum samples were tested in duplicate. The amount of antigen specific IgE was also determined using a similar ELISA protocol using the secondary rat anti-mouse IgE HRP antibody (Southern Biotech). Total IgE was determined using GenWay’s mouse IgE kit. The manufacturer’s protocol was followed with serum dilutions at 1:50. All serum samples were tested in duplicate. Methods References 1. Yan J, Yoon H, Kumar S, Ramanathan MP, Corbitt N, Kutzler M, et al. Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Molecular Therapy 2007;15:411-412. 2. Yan J, Yoon H, Kumar S, Ramananthan MP, Corbitt N, Kutzler M, et al., Enhanced diversity and magnitude of cellular immune responses elicited by a novel engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Molecular Therapy 2007;15:411-21 3. Shedlock DJ, Aviles J, Talbott KT, Wong G, Wu SJ, Villarreal DO, et al., Induction of broad cytotoxic T cells by protective DNA vaccination against marburg and ebola. Mol Ther 2013;21:1432. 4. Wherry EJ, Blattman JN, Murali-Krishna K, van der Most R, Ahmed R. Viral persistence alters CD8 T-cell immunodominance and tissue distrubution and results in distinct stages of functional impairment. J Virol 2003;77:4911-27. 5. Angelosnato JM, Blackburn SD, Crawford A, Wherry EJ. Progressive Loss of Memory T cell potential and commitment to exhuastion during chronic viral infection. J Virol 2012;86:8161-70.