the latter. It has been reported that mature DC continue to employ receptor-mediated endocytosis upon maturation [30], despite macropinocytosis being significantly reduced. Such ob- servations demonstrate that macropinocytosis may be the major route for endocytosis of SVLP by DC, but other endocytic processes, albeit less efficient, can be involved. Regardless, internalisation of SVLP by DC is a gradual process, typical of what has been reported for DC antigen processing [9]. The present work sought to characterize how DC interact with a complex, virus-like antigenic structure – SVLP – which is an efficacious immunogen requiring no adjuvant to induce adaptive immunity in mice, rabbits [17] and pigs (R.S., unpublished data). It was considered that this interaction would highlight the elements contributing to efficient handling by DC leading to the observed in vivo efficacy. Overall, DC interacted rapidly with the SVLP, but displayed a more gradual kinetics for uptake and processing, the latter relating to reported in vivo characteristics for DC [9]. Endocytosis was a relatively slow process dominated by caveolin-independent, lipid raft polarisation, leading the SVLP into a peripheral location. The processing was dominated by macropinocytosis, although non-macropinocytic events were likely in the background. The processing was characterized by slow kinetics, involving fusion with EEA-1+ early endosomes in the periphery, and followed a common path to that with dextran. This did not lead into the more degradative pathway employed with transferrin, nor was there any observable association with lysosomes. The rare localisation with MHCII-structures and the ER may reflect a loss of signal once the material reached these organelles, due to proteolytic cleavage. It is also possible that such rare localisation reflected a slow progress of material into these structures, at a level too low to be detected. The prolonged association with peripheral structures, which were not all EEA-1+, and the slow processing relate to macropinosomal retention of antigen for slow antigen processing [9], and would allow for entry into sorting compartments favouring transfer of antigen to B lymphocytes [1,29]. Certainly, the processing was effective, as witnessed by the immune responses induced by SVLP in vivo. These characteristics of how DC handle a complex virus-like structure such as SVLP do relate to efficacious processing, and the characteristics DC require for efficient induction of immune responses. Materials and Methods Ethics Statement Blood donor pigs were used for the supply of blood, from which blood cells were prepared, and used for the generation of monocyte-derived DC. This operated under permission from the Canton of Bern, Switzerland through the animal licence 112/09. Porcine monocyte-derived DC Porcine monocyte-derived DC were prepared as previously described [31], from specific pathogen-free pigs held at the Institute’s facility. Briefly, these were prepared from peripheral blood cells using CD172a-magnetic sorting to isolate monocytes, then culturing for 3–5 days with recombinant porcine GM_CSF and IL-4 as described [32]. Monocytes are characterised by high expression of CD172a (Fig. S1C), and the sorting yields .90% purity. Upon culture in GM-CSF and IL-4, we obtained $95% DC purity, characterized as described [32], [31]. Cell culture medium and serum was obtained from Invitrogen (Basel, Switzer- land). DC Endocytosis of SVLP SVLP Lipopeptide buildings blocks were prepared as described previously [17], Briefly, the peptide GGIEKKIEAIEKKIEAIEK- KIEAIEKKIEAIEKKIAKMEKASSVFNVVNSKKKC-DA-am- ide (0.25 mmol scale) was assembled on an ABI 433A peptide synthesizer using standard Fmoc chemistry and Rink amide MBHA resin pre-loaded with Fmoc-D-Ala (substitution: 0.3 mmol/g). After assembly and removal of the N-terminal Fmoc-protecting group using 20% piperidine in DMF, the lipid 1,3-dipalmitoyl-glycero-2-phosphoethanolamine-N-succinic acid [17] was coupled manually using PyBOP/HOBt/DIEA for activation. The resin was then washed with DMF (3x), CH2Cl2 (3x) and CH3OH (3x), dried in vacuo over KOH pellets and treated with CF3COOH/thioanisole/ethanedithiol/H2O/i- Pr3SiH (75:10:10:4:1, 10 ml). The lipopeptide was precipitated with iPr2O (cooled to 220u), washed 3x with iPr2O, dried and purified by reverse phase HPLC using an UP10WC4/25M preparative C4 column (Interchrom, 250621.2 mm, 5 mm parti- cle size, 300 A˚ pore size). For cell-uptake and intracellular trafficking studies, SVLP were labelled with Alexa-Fluor 488 C5 maleimide (Life Technologies, Zug, Switzerland). Remaining free cysteines were blocked with N-ethylmaleimide (Sigma). For some experiments, in order to observe degradation in acidifying compartments, over-labelled SVLP were prepared by conjugating excess Alexa-Fluor 488 C5 maleimide. The molecular weights of the lipopeptide and Alexafluor 488 conjugate were confirmed by mass spectrometry (MALDI-TOF and/or ESI-MS) and the purity was .96% by analytical reversed phase HPLC. The size distribution of SVLP was monomodal with narrow size-dispersion and a hydrodynamic radius [14] of 11.2–12.8 nm by dynamic light scattering (DLS). Unless stated otherwise, the SVLP were used at 1 mg/ml for Flow Cytometry and 2.5 mg/ml for confocal microscopy. Proteinase K and pronase treatment of DC In order to quantify SVLP internalisation by DC, flow cytometry was employed. To this end, it was important to differentiate between the surface bound and internalised SVLP. Accordingly, Proteinase K (PK) obtained from Sigma was employed. PK was titrated with DC on ice, to determine the least toxic concentration, PK induced toxicity being observed by PI staining. Least toxicity was seen with 0.2 mg/ml of PK. Therefore, 0.2 mg/ml of PK was used in all the experiments. PK was always used under cold conditions on ice in order to minimise cell damage. Pre-chilled DC were incubated with SVLP on ice for 20 or 30 min, or without SVLP were washed with 2 ml ice cold phosphate-buffered saline (PBS). Cell pellet was re-suspended in 500 ml of cold Dulbecco’s MEM (DMEM) and incubated on ice for 20 min. Then PK (0.2 mg/ml) was added followed by 30 min incubation on ice. Then, 10% (v/v) porcine serum (PS) was added in order to inactivate PK activity. Cells were then washed with 2 ml ice cold cell wash or PBS by centrifuging at 3506 g. Cells were re-suspended in cell wash to analyse by flow cytometry or in DMEM to incubate with SVLP. DC were also treated with pronase (Roche), for stripping protein receptors. DC (0.56106/ml) were treated with different concentrations of pronase and incubated at 39uC for 25 min, followed by 5 times washing with 2 ml ice cold cell wash containing 10% (v/v) PS. DC were immediately re-suspended in 500 ml cold DMEM and shifted on ice for 30 min, followed by addition of SVLP or CTB and incubated for 20 min on ice. Cells were then fixed with 4% (w/v) PFA, or surface labelled with antibody against CD172a, CD14 or MHCI; replicates of cells PLOS ONE | www.plosone.org 14 August 2012 | Volume 7 | Issue 8 | e43248