To differentiate monocytes into immature DCs 250 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF) and 100 U/ml IL-4 (Invitrogen) was IOX1 clinical trial added. Medium was refreshed after 3 days. DC were incubated for 48 h at 37 °C in RPMI 1640 containing 500 U/ml GM-CSF with OVA (highest

concentration 5 μg/ml), either free or encapsulated into liposomes with and without PAM or CpG (highest concentration 10 μg/ml), keeping the lipid:OVA:TLR ligand ratio 50:2:1 (w/w). OVA, OVA liposomes and mixtures of OVA with PAM or OVA with CpG were used as controls and LPS (100 ng/ml, Invivogen) was added as a positive control. Cells were washed 3 times with PBS containing 1% (w/v) bovine serum albumin and 2% (v/v) FCS and incubated for 30 min with a mixture of 20× diluted anti-HLADR-FITC, anti-CD83-PE and anti-CD86-APC (Becton Dickinson) in the dark at 4 °C. Cells were washed and the expression of MHCII, CD83 and CD86 was quantified using flow cytometry (FACSCanto II, Becton Dickinson) relative to LPS, assuming 100% maturation for LPS-treated DC. Live cells were gated based on forward and side scatter. Groups of 8 mice were immunised with the OVA-loaded liposomes with and without PAM or CpG by ID injection into the abdominal skin as described

previously [30]. Besides the liposomes, solutions of OVA or OVA with PAM or CpG in PBS were injected and subcutaneous (SC) injection of OVA served as a control. The mice were vaccinated twice with three weeks intervals

with a dose of 5 μg learn more OVA and 10 μg PAM or CpG in a total volume of 30 μl. To maintain this either ratio between antigen and immune potentiator, liposomes used for the immunisation study were not filtered to remove free antigen and TLR ligand. Blood samples were collected from the tail vein 1 day before each immunisation. Three weeks after the last vaccination the mice were sacrificed. Just before euthanasia total blood was collected from the femoral artery. Afterwards the spleens were removed. Blood samples were collected in MiniCollect® tubes (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) till clot formation and centrifuged 10 min at 10,000 × g to obtain cell-free sera. The sera were stored at −80 °C until further use. OVA specific antibodies (IgG, IgG1 and IgG2a) in the sera were determined by sandwich ELISA as described previously [30]. Briefly, plates were coated overnight with 100 ng OVA/well. After blocking, two-fold serial dilutions of sera from individual mice were applied to the plates. HRP-conjugated antibodies against IgG, IgG1 or IgG2a were added and detected by TMB. Antibody titres were expressed as the reciprocal of the sample dilution that corresponds to half of the maximum absorbance at 450 nm of a complete s-shaped absorbance-log dilution curve.