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Blood, Vol. 113, Issue 15, 3520-3529, April 9, 2009
Viperin is required for optimal Th2 responses and T-cell receptor–mediated activation of NF- B and AP-1
Blood Qiu et al.
113: 3520
Supplemental materials for: Qiu et al
Immunofluorescent staining and confocal microscopy
Stimulated cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin, and then stained with respective primary antibodies and Alexa Fluor (AF) 488- and AF 594-conjugated secondary antibodies (Molecular Probes). Cryosections of spleens were fixed with methanol and stained with antibodies against viperin and CD marker where indicated, after blocking in 10% goat serum for 30 min. This was followed by detection with AF 488-conjugated goat anti-rabbit IgG and AF594-conjugated goat anti-rat IgG, and then visualized with a LSM 510 Meta confocal microscope (Zeiss, Jena, Germany) equipped with 40 ×/0.75 and 100 ×/1.3 oil Plan-Neofluor objective lens. Flow cytometry
Single-cell suspensions of spleens were prepared and 1 × 106 cells were stained with the respective antibodies for 30 min at 4°C after preincubation with anti-FcγR for 10 min. FITC-, PE-, or APC-conjugated antibodies against CD3 (145-2C11), CD4 (RM4-5, GK1.5), CD8a (53-6.7), CD19 (1D3), CD44 (IM7), CD62L (MEL-14), CD25 (IL-2Rα, PC61) and CD69 (H1.2F3), were from BD PharMingen. Expression of CD124 (IL-4Rα) and CD132 (common γ chain, γc) was determined using biotin-conjugated anti-CD124 or anti-CD132 antibody (PharMingen) followed by PE-conjugated strepavidin (Jackson ImmunoResearch). Stained cells was acquired on a FACSCaliber flow cytometer (Becton Dickinson) using CellQuest and analyzed with FlowJo analysis software (Tree Star). Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. Immunization and serum immunoglobulin measurements
Six-week-old female mice were i.p. immunized with 100 µg of ovalbumin (OVA, grade V, Sigma) emulsified in complete Freund adjuvant (CFA), followed by a second injection of the same quantity of OVA, mixed with IFA, at two weeks after primary immunization. Mice were bled before and after injection on a weekly basis, and sera were collected to determine the concentrations of OVA-specific IgG1, IgE and IgG2a using ELISA plates that were coated with 10–100 µg/ml ovalbumin in PBS. ELISA detection of antigen-specific immunoglobulins (Igs) and basal levels of different Ig subclasses were analyzed using isotype-specific antibodies with a standard sandwich ELISA protocol. Purified isotype-specific mouse Ig subclasses (PharMingen) were used as standards. Cytokine measurements
Sandwich ELISAs for IL-2, IL-4, IL-5, and IFN-γ were performed using the OptEIA Mouse kit (BD Biosciences) according to the manufacture’s protocol. The production of IL-13 was measured with mouse IL-13 ELISA kit (R&D Systems) following the manufacturer’s instructions. Calcium mobilization
Freshly isolated splenic CD4+ T cells were resuspended at 2 × 106/ml in RPMI 1640 medium containing 2% FCS and 10 mM HEPES, and loaded with 1 µg/ml of Indo-1 (Molecular Probes). Crosslinking was initiated by adding anti-CD3 and goat anti-hamster IgG (Jackson ImmunoResearch) sequentially, followed by ionomycin to induce the full scale deflection of calcium flux. The ratio of bound Indo-1 fluorescence to unbound Indo-1 fluorescence was recorded by a LSR II flow cytometer (Beckton Dickinson), and data were analyzed with FlowJo cytomtetric analysis software. Quantitative transcript analysis
RNA was isolated using the QIAGEN RNeasy miniKit according to the manufacturer’s protocol and residual genomic DNA was removed by incubating with RNase-free Dnase I (Qiagen). First-strand cDNAs were synthesized using oligo(dT)12–18 primers and SuperScript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed using SYBR Green and the ABI Prism 7900 Sequence Detection System (Applied Biosystems). Results were normalized with β-actin and then used to calculate expression levels according to the ΔΔCt method.1 All data were expressed relative to the unstimulated or anti–CD3-treated wild-type sample which was set as one. Primers were designed and blasted against mouse genome to ensure that no cross-reactivity with other genes would occur. The real-time PCR primers are available upon request. REFERENCE 1. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29: e45.
Files in this Data Supplement:
- Figure S1. Kinetic analysis of viperin mRNA expression in response to anti-CD3 with or without anti-CD28 stimulation at different time points (JPG, 16.5 KB)
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A representative of 3 independent experiments is shown. Data are expressed as the mean value of duplicate-triplicate determinations ± SD. **P<0.01, 72 hr anti-CD3 ± CD28 treatment vs. 36 hr corresponding stimulation.
- Figure S2. Thymocyte development was not perturbed in viperin-deficient mice (JPG, 48.7 KB)
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(A) CD8/CD4 profiles of thymocytes from 6-week-old wild-type and viperin−∕− mice. The numbers in quadrants indicate the percentage of each quadrant. Data are representative of four independent experiments; (B) Mean cell numbers (± SD) for each subpopulation, determined by multiplying the percentage of cells in each subset by the total number of thymocytes and averaging 3–4 mice per genotype. DP, double-positive; DN, double-negative; SP, single-positive.
- Figure S3. Surface expression of CD44 and CD62L on splenic CD8+ T-cell population from viperin−∕− mice and wild-type controls (JPG, 41.5 KB)
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Data are representative of three independent experiments.
- Figure S4. Functional characterization of viperin−∕− mice (JPG, 32.8 KB)
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(A) Basal levels of immunoglobulins (Igs) in serum samples from 6-week-old wild-type (●) and viperin−∕− mice (○) (n=8 and 16, respectively). Mean concentration for each genotype is indicated (horizontal line); (B) Wild-type (△), heterozygous (■), and homozygous (♦) mice were immunized with OVA/CFA at day 0 and with OVA/IFA at day 14. Production of OVA-specific IgG1 in immunized mice was analyzed. Data are from two independent experiments, and mean concentration of OVA-IgG1 from a total of 9–11 mice in each genotype group is shown. Statistical significance was determined using 2-tailed Student t test, *P=0.014 WT vs. KO.
- Figure S5. Immunoblotting analyses of Th2-associated transcription factors in total splenic CD4+ T cells in the presence of anti–IL-4 antibody 10 µg/ml after 48 hr culture (JPG, 28.5 KB)
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Data are representative of two independent experiments. A vertical line has been inserted to indicate a repositioned gel lane.
- Figure S6. Normal production of Th2 cytokines by viperin-deficient CD4+ memory T cells after stimulation with anti-CD3 (10 µg/ml) ± anti-CD28 (5 µg/ml) Abs for 72 hr (JPG, 31 KB)
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Results are expressed as the mean value of triplicate determinations±SD. A representative of three independent experiments is shown.
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