Both CLR-mediated and CD4-mediated endocytosis of Env result in similar kinetics of antigen processing Cells were incubated with 10µg/mL FITC-labeled Env or HA for 90 min at 4°C or 37°C, then washed twice and cultured at 4°C or 37°C for up to 24h. In some cases, MDCs were pre-incubated and the experiment carried out in the presence of 1mM leupeptin (Leu) to inhibit proteolytic degradation. Samples were then stained with DC markers for analysis by flow cytometry and gated on HLA-DR + /CD11c + (MDCs), CD123 + (PDCs), CD1a + (LCs) or HLA-DR + (DDCs). A) Representative MDC donor showing the percentage of positive cells. B) The MFI for each timepoint was normalised to the 0 min 37°C control for each Ag and the data shown here are the mean ± SD for for 3–5 donors for Env and 1 donor for HA. C) Alternatively, MDCs were incubated with Env in the presence of mannan or incubated with Env-D368R to isolate degradation via CD4 and CLR-mediated uptake pathways respectively. Leupeptin was also included in these experiments as a control. Data here are the mean ± SD of 2 donors.
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