6-10-week-old C57BL/6 (B6) mice and OT II transgenic mice were purchased from the Jackson Laboratory Bar Harbor, ME). TCRδ−/− mice were a kind gift from Dr. E. ( Fikrig (Yale University, New Haven). Groups were age and sex-matched for each experiment and were housed under identical conditions. All animal experiments were approved by the Institutional Animal Care and Use Committee at Colorado State University.
Infection in mice
WNV NY99-6480 was passaged three times in C6/36 Aedes albopictus cells to make a virus stock for both cell culture and in vivo studies. Mice were inoculated intraperitoneally with 100 PFU of WNV NY99-6480 isolate.
Purification of DCs, CD4+, and γδ T cell subsets
DCs, CD4+ and γδ T cells were purified from pooled spleens of 3–5 mice by a positive selection method, using anti-CD11c, anti-mPDCA-1, anti-CD4 magnetic beads or FITC-conjugated anti-mouse TCRγδ BD Biosciences, San Diego, CA) followed by anti-FITC magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The purity of these cells is 82–95%. For FACS purification of γδ T cells, splenocytes were enriched for Tcells using anti-CD90 magnetic beads (Miltenyi Biotec), stained with FITC labeled anti-TCRγδ and sorted based on expression of TCRγδ (MoFlo, DakoCytomation). The purity of γδ T cells was 94.3%.
Antibodies for CD40, CD80, CD86, I-Ab and CD11c were purchased from BD Biosciences. Following staining, cells were fixed in PBS with 1% paraformaldehyde and examined using a Cyan flow cytometer (Dako Cytomation). Data were analyzed using Summit 4 software (Dako Cytomation). For intracellular cytokine staining, splenocytes were stimulated at 2 × 106 cells/well with 10μg/ml LPS (Sigma-Aldrich, St. Louis, MO) or 0.5 μg/ml CL097 (Invivogen, San Diego, CA) with Golgi-Plug for 4 h at 37°C. Cells were harvested, stained with FITC-anti-CD11c, fixed in Fixation/Permeabilization solution before adding PE- anti-IL-12 (eBioscience, San Diego, CA) or rat IgG2a (BD Biosciences).
In vitro DC maturation and T cell priming assays
Naïve DCs were co-cultured with γδ T cells from non-infected or WNV-infected mice at 1: 1 ratio in 24-well plates at 37 °C for 24 h. Cells were harvested and stained with antibodies for cell surface markers. CD11c+ cells were gated for analysis. DCs were also co-cultured with in vitro WNV-infected γδ T cells. CD4+ T Cells and DCs were purified from splenocytes of OT II transgenic mice or WNV-infected mice at day 3 post-infection. CD4+ T Cells (1 × 105) were mixed with DCs at 1:1 ratio and treated with or without OVA residue 323–339 (10μg/ml, Genscript Corporation, Piscataway, NJ).
WNV infection or stimulation with TLR agonist in γδ T cells
γδ T cells (1 × 105 cells/well) were cultured for 2 days at 37 °C in RPMI-1640 medium (Invitrogen, Carlsbad, CA) in 96-well plates coated with 5μg/ml anti-CD3 (eBioscience). Cells were infected with WNV at a MOI of 0.5 for 1 h, washed and incubated in the above medium containing 5 ng/ml recombinant human IL-2 (eBioscience). H36.12j cells (macrophage cell line, American Type Culture Collection, Manassas, VA) were infected with WNV (MOI = 1) and harvested at day 4 post-infection. In some experiments, γδ T cells were stimulated with poly I: C (Amersham Pharmacia, New Jersey, 50μg/ml) or CL097 (Invivogen, 0.5μg/ml).
Quantitative PCR (Q-PCR) and PCR for determining viral load and TLR levels
RNA was extracted using RNAeasy extraction kit (Qiagen, Valencia, CA) and was used to synthesize complementary (c)DNA using the ProSTAR First-strand RT-PCR kit (Stratagene, Cedar Creek, TX). The sequences of the primersets for WNV envelope gene (WNE
), Tlr2, Tlr3, Tlr4
cDNA and PCR conditions were described previously (Lanciotti, et al., 2000
, Schulz, et al., 2005
, Chen, et al., 2006
). Q-PCR analysis was performed with RT2 Real-Time SYBR Green / Fluorescein PCR master mix (Superarray, Frederick, MD) on an iCycler (Bio- Rad, Hercules, CA). The ratioof the amount of amplified gene compared with the amount of β
cDNA represented the relative levels in each sample. Regular PCR was performed as follows: 94°C for 2 min followed by 35 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1.5 min, and final extension at 72°C for 5 min. The primer pairs used were: TLR2, 5’-CAG ACG TAG TGA GCG AGC TG-3’ and 5’-GGC ATC GGA TGA AAA GTG TT-3’; TLR3, 5’-CCC CCT TTG AAC TCC TCT TC-3’ and 5’-TTT CGG CTT CTT TTG ATG CT -3’; TLR4, 5’-GCT TTC ACC TCT GCC TTC AC-3’ and 5’-CGA GGC TTT TCC ATC CAA TA-3’; TLR7, 5’-CAT CAG AGG CTC CTG GAT GA-3’ and 5’-AGG CAT GTC CTA GGT GGT GA-3’. The sequences of the primersets for β-actin
were described earlier (Farrar & Street, 1995
Fluorescence microscopy with γδ T-cells
Cells were fixed in acetone at −20°C for 30 min, rehydrated in PBS and stained with FITC-conjugated anti-CD3, Phycoerythrin (PE)-conjugated anti-TCRβ (eBioscience) and the flavivirus E protein-specific monoclonal antibody 4G2 followed by Alexa Fluor 350-conjugated anti-mouse IgG2a (Invitrogen) at 37°C. Images were acquired on an Olympus IX71 Inverted Microscope at 20 × magnification.
Vero cells were seeded in DMEM (Invitrogen) with 10% FBS and incubated with serial dilutions of culture supernatant for 2 h. DMEM containing 5% FBS and 1% low-melting-point agarose was added and incubated for 4 days. A second overlay of 1% agarose-medium containing 0.01% neutral red was added to visualize plaques.
Cytometric bead array and ELISA
Culture supernatant was measured for cytokine production using a mouse Th1/Th2 kit or an inflammation kit by a FACSArray analyzer (BD Biosciences). Cytokine levels were also measured by ELISAs (BD Biosciences & PBL Interferon Source).
Data analysis was performed using Prism software (Graph-Pad) statistical analysis. Values for phenotype analysis, viral burden, and cytokine production experiments were presented as means ± SEM. P values of these experiments were calculated with a non-paired Student’st test or Mann-Whitney test. Statistical significance was accepted at P < 0.05.