Plasmacytoid dendritic cells (pDCs) play a crucial role in initiating immune responses by secreting large amounts of type I IFNs. Currently, the role for human pDCs as professional APCs in the cross-presentation of exogenous Ags is being re-evaluated. Human pDCs are equipped with a broad repertoire of Ag uptake receptors and an efficient Ag-processing machinery. In this study, we set out to investigate which receptor can best be deployed to deliver Ag to pDCs for Ag (cross-)presentation. We show that targeting nanoparticles to pDCs via the C-type lectins DEC-205, DC immunoreceptor, blood DC Ag-2, or the FcR CD32 led to uptake, processing, and (cross-) presentation of encapsulated Ag to both CD4+ and CD8+ T cells. This makes these receptors good candidates for potential in vivo targeting of pDCs by nanocarriers. Notably, the coencapsulated TLR7 agonist R848 efficiently activated pDCs, resulting in phenotypical maturation as well as robust IFN-α and TNF-α production. Taken together, their cross-presentation capacity and type I IFN production to further activate components of both the innate and adaptive immune system mark pDCs as inducers of potent antitumor responses. These findings pave the way to actively recruit human pDCs for cellular cancer immunotherapy.

To address how surface charge affects the fate of potential nanocarriers in the lung, gold nanoparticles (AuNPs) coated with polyvinyl alcohol containing either positively (NH) or negatively (COOH) charged functional groups were intra-nasally instilled in mice, and their uptake by antigen presenting cell populations (APC) in broncho-alveolar lavage (BAL) fluid, trachea, and lung parenchyma, as well as trafficking to the lung draining lymph nodes (LDLNs) was assessed by flow cytometry. Furthermore, CD4 T cell proliferation in LDLNs was investigated following instillation. All APC subpopulations preferentially captured positively-charged AuNPs compared to their negatively-charged counterparts. Uptake of AuNPs up-regulated expression of co-stimulatory molecules on all APC populations. Furthermore, positively-charged AuNPs induced enhanced OVA-specific CD4 T cell stimulation in LDLNs compared to negatively-charged AuNPs, or polymer alone. Our findings demonstrate surface charge as a key parameter determining particle uptake by APC, and down-stream immune responses depend on the presence of particle core-bound polymer.

The respiratory tract with its ease of access, vast surface area and dense network of antigen-presenting cells (APCs) represents an ideal target for immune-modulation. Bio-mimetic nanocarriers such as virosomes may provide immunomodulatory properties to treat diseases such as allergic asthma. In our study we employed a triple co-culture model of epithelial cells, macrophages and dendritic cells to simulate the human airway barrier. The epithelial cell line 16HBE was grown on inserts and supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) for exposure to influenza virosomes and liposomes. Additionally, primary human nasal epithelial cells (PHNEC) and EpCAM+ epithelial progenitor cell mono-cultures were utilized to simulate epithelium from large and smaller airways, respectively. To assess particle uptake and phenotype change, cell cultures were analyzed by flow cytometry and pro-inflammatory cytokine concentrations were measured by ELISA. All cell types internalized virosomes more efficiently than liposomes in both mono- and co-cultures. APCs like MDMs and MDDCs showed the highest uptake capacity. Virosome and liposome treatment caused a moderate degree of activation in MDDCs from mono-cultures and induced an increased cytokine production in co-cultures. In epithelial cells, virosome uptake was increased compared to liposomes in both mono- and co-cultures with EpCAM+ epithelial progenitor cells showing highest uptake capacity. In conclusion, all cell types successfully internalized both nanocarriers with virosomes being taken up by a higher proportion of cells and at a higher rate inducing limited activation of MDDCs. Thus virosomes may represent ideal carrier antigen systems to modulate mucosal immune responses in the respiratory tract without causing excessive inflammatory changes.