Kimura et al

SUPPLEMENTAL MATERIAL

MATERIAL AND METHODS:

Confocal Microscopy Analyses of GFP/ MHCII Expression. Spleen and liver specimens were dissected immediately after CO2 euthanasia, embedded in optimal cutting temperature compound (OCT, Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. Cryostat sections (5-7 mm) were fixed in 4% cold paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PBS) at 4°C for 15 min, rinsed with PBS 3 times for 5 min, pre-incubated with 1% goat serum (diluted in PBS with 0.1% Triton-X-100) for 1 hour at room temperature, and rinsed with PBS (3x , 5 min). Sections were incubated with rat anti-mouse I-Ab (MHCII) monoclonal antibody (AF6-120.1, Pharmingen, 1:200 in 0.1% Triton-X-100/PBS), incubated for 1 h at room temperature, washed with PBS (3x, 5 min), then incubated with a mixture of rabbit anti-GFP IgG conjugated with Alexa 488 (Molecular Probes, diluted 1:1000) and goat anti-rat IgG conjugated with Alexa 555 (Molecular Probes diluted 1:200 in blocking buffer) and incubated for 1h at room temperature in the dark. Sections were washed with PBS (3x, 5 min), nuclei were counterstained with TOPRO-3 (Molecular probes, diluted 1:1000 in PBS) for 5 min and slides were coverslipped. Fluorescent images were prepared using the indirect immunofluorescence technique and acquired with a Zeiss Confocal Laser Scanning Microscope LSM 510 META (Zeiss, Thronwood, NY), equipped with a Plan Apochromat 63x (1.4 NA) oil objective. The images were acquired as 12 bit signals with a resolution of 1024 x 1024 pixels. To identify fluorescent signals different lasers were used for excitation, for Alexa 488 (GFP) the 488 nm argon laser line was used, for Alexa 555 (MHCII) the 543 nm HeNe laser line was used, and for TOPRO3 (nuclei) the 633 nm HeNe laser line was used. Acquisition of emission signals was collected sequentially to prevent spectral bleedthrough. Specific bandpass filters were used to achieve proper separation of emission signals (488/505-530BP, 543/560-610BP, 633/650LP). All post image processing was performed using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA).

RT-Q-PCR Analysis of Genomic DNA: Quantification of the vector copy number in the was performed in 25 ml reaction containing 300 ng of genomic DNA (equivalent to 5 x 104 genomes) using TaqMan RT-Q-PCR assay to detect lentiviral packaging sequences of HIV1 as a universal primer [42]. The oligonucleotides used were: LV primers: 5’-ACCTGAAAGCGAAAGGGAAAC-3’ and 5’-CACCCATCTCTCTCCTTCTAGCC-3’; LV probe: 5’-FAM-AGCTCTCTCGACGCAGGACTCGGC-TAMRA-3’; beta-actin internal control primers: 5’-GGTCGTACCACAGGCATTGT-3’, and 5’-CTCGTAGATGGGCACAGTGT-3’ and beta-actin probe: 5’-FAM-CCCGTCTCCGGAGTCC-NFQ-3’. Amplifications were carried out in an ABI PRISM 7700 sequence detector (Perkin Elmer, Wellesley, Massachusetts); after the initial denaturation (10 min at 95 ºC), amplification was performed with 40 cycles of 15 s at 95 ºC and 60 s at 60 ºC. To calculate the copy number in the samples, a reference curve was prepared by amplifying serial dilutions of LV-CMV-LUC plasmid in a background of genomic DNA of spleen from non-treated mice; the Ct values were plotted against the input plasmid, and a standard reference curve was obtained.

In vivo Bioluminescence Imaging Analyses: Mice were anesthetized intraperitoneally with ketamine (100 mg/kg) and xylazine (10 mg/kg), and an aqueous solution of D-luciferin (150 mg/kg) was injected intraperitoneally 10 minutes prior to imaging. Animals were placed into the light chamber of the CCD camera (IVIS, Xenogen, Alameda, CA), and grayscale body surface reference images (digital photograph) were taken under weak illumination. After switching off the light source, photons emitted from luciferase-expressing cells within the animal body and transmitted through the tissue were quantified for 2 minutes using the software program “Living Image” (Xenogen) as an overlay on Igor (Wavemetrics, Seattle, WA). For anatomical localization, a pseudocolor image representing light intensity (blue, least intense; red, most intense) was generated in “Living Image” and superimposed over the grayscale reference image. For quantitation of transmitted light, a region of interest (ROI) was manually selected over the signal intensity. The intensity was recorded as maximum (photons/cm2/sr) within a ROI. To investigate the statistical change in the bioluminescence signal intensity during the study period, outcome by time scatter plot with a Loess regression curve was produced for each group. Based on the plots, a first-degree spline model was developed. This model fitted line pieces for weeks 1-8 and 8-39, and these pieces were joined together at week 8 to achieve continuity. Slopes of these lines were estimated and compared between the LV-CMV-LUC and LV-MHCII-LUC using SAS Proc Mixed procedure. A random intercept was set in the model to allow the difference among subjects. Since the slopes were not significantly different between Weeks 1-8 and 8-39 for the C57BL/6 mouse group, one line was fitted for each treatment in this group.

Intracellular IFN-g staining: splenocytes were cultured for 6 hrs in 96-well plates at a concentration of 1 x 106 cells/well in 0.2 ml of complete medium with 1 µl/ml brefeldin A (GolgiPlug; PharMingen) and human IL-2 (50 U/ml). The cells were restimulated with TRP2 (TRP2: 180-188, SVYDFFVWL) or the unrelated lymphocytic choriomeningitis virus nucleoprotein (LCMV NP: 396-404, FQPQNGQFI) peptides at 5 mg/ml. The cells were spun down and surface antigens were stained in PBS, supplemented with 2% FBS with PE-conjugated MAb for CD8 and PerCP-conjugated MAb for CD3. After washing off the unbound antibody, cells were subjected to intracellular cytokine stain using the Cytofix/Cytoperm kit (PharMingen) according to manufacturer’s instructions. For intracellular IFN- staining, the FITC-conjugated monoclonal rat anti-mouse IFN-g Ab (clone XMG1.2; PharMingen) or its isotype control Ab (rat IgG1) were used. Flow cytometry was performed with a Beckman/Coulter flow cytometry apparatus. For each set of analyses, 5x104 cells were acquired. To establish background for fluorescence and to set gates for data acquisition, cells stained with isotype antibodies were used as baseline reference. Care was taken to analyze cells that were in the viable white blood cell gate as indicated by forward and side scatter characteristics.

LEGENDS

Supplemental Figure 1: Quantitative flow cytometry analyses of spleen. 100,000 viable cells were gated as R1 in forward and side scatter plots. Cells positive for each PE-immunostaining staining were gated as R2, using the isotype control antibody as reference. R3 represents the GFP+ fraction of R2. Frequency of GFP+ cells for each cell sub-population (in %) and mean fluorescence intensity (MFI) for the gated GFP+ cells are shown.

Supplemental Figure 2: Diagrammatic representation of hypothetical mechanisms by which LVs with CMV or MHCII promoter after intravenous administration might present with different outcomes in immune stimulation (LV-CMV) versus persistence of transgene expression and viral integration (LV-MHCII).

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