BafA prevents the concentration of imiquimod in the MIIC. pDCs, precultured with IL-3 for 24 hours, were treated or not with BafA (100nM) for 2 hours. Then, cells were stimulated with R837-TAMRA at 50 μm (A) or CpG-B–TAMRA at 10 μm (B) for the indicated times at 37°C or at 4°C. Cells were stained with Live/Dead and analyzed by flow cytometry for compound uptake gating on live cells. The mean fluorescence of cells alone cultured under the same conditions was subtracted (δ mean fluorescence). Cell viability was not affected by BafA treatment. Data were obtained with pDCs purified from a single donor and were reproduced with pDCs from 2 other donors. (C) Model for the concentration of TLR7 agonist small molecules in acidic endosomes on the basis of the previous data. R837 is a cell-permeable weak base that passively diffuses everywhere inside the cell (fast phase, v-ATPase-independent) and then starts to accumulate in the class II loading compartment where it gets protonated and therefore trapped (slow phase, v-ATPase-dependent). In the presence of BafA, v-ATPase is blocked, and endosomal pH is no longer acidic. Therefore, R837 does not get protonated in endosomes and does not accumulate there.