Download Supplementary Materials and Methods Transfection and expression

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.