Modified Vaccinia Virus Ankara Induces Toll-Like Receptor-Independent Type I Interferon Responses

Cell isolation and culture

BM cells were isolated by flushing femur（股骨） and tibia（胫骨）with RPMI medium supplemented with 10% fetal calf serum（胎牛血清）.

Upon red blood cell lysis（溶解）, cells were washed and seeded at a density of 1×10⁶ cells/ml or 2× 10⁶ cells/ml in medium supplemented with GM-CSF (100 ng/ml; R&D Systems) or Flt3-L  (100  ng/ml;  R&D  systems),  respectively. 

Flt3-L-supplemented  cultures (BM-derived  pDC  [BM-pDC])  were  cultivated  for  8  days  with  one  medium
change  at  day  4,  whereas  medium  of  GM-CSF-supplemented  cultures  (BM-derived mDC [BM-mDC]) was changed every 1 to 2 days, depending on the status  of  cultures,  by  replacing  one-half  of  the  medium  with  fresh  cytokine-supplemented medium.

Flow cytometric analysis and cell enrichment.

Cells were stained with anti-B220-phycoerythrin (PE) or -PECy5.5 monoclonal antibody (MAb), anti-CD11c-allo-phycocyanin MAb, anti-CD69-PE MAb, or anti-CD86-fluorescein isothiocyanate MAb (all from BD PharMingen).

For detection of VACV-specific surface proteins, VIG was used in combination with F(ab)2-PE.

For enrichment of CD11c⁺ B220⁺  cells from Flt3-L BM cultures, MS or LD columns (Miltenyi Biotech)
were  used  according  to  the  manufacturer’s  instructions. 

The  purity  of  pDC (CD11c⁺ B220⁺) usually exceeded 80%.

In vitro stimulations and quantification of cytokine production.

For stimulation experiments, ex vivo-isolated BM cells or in vitro-differentiated bulk cultures of BM-mDC and bulk cultures of BM-pDC were seeded at 1 × 10⁶ cells/well in 24-well  culture  plates  in  1  ml  medium.  Magnetically  activated  cell  sorting (MACS)-sorted cells were seeded at a density of 2 ×10⁵  cells/well in 96-well
culture  plates  in  200  μl  medium.  CpG  2216  (ggGGGACGATCGTCgggggG[lowercase indicates phosphorothioate-modified nucleotides]; Sigma-ARK) was used at a final concentration of 10 μg/ml. For transfection of 2 g poly(I:C) (Sigma-Aldrich), the reagent Fugene (Roche) was used according to the manu-
facturer’s instructions. After stimulation, cell-free supernatant was collected and analyzed with a mouse IFN- or mouse IFN- enzyme-linked immunosorbent assay (ELISA) kit (PBL Biomedical Laboratories).