Differential cytosolic delivery and presentation of antigen by listeriolysin O-liposomes to macrophages and dendritic cells.

Delivery of antigenic protein to the cytosol of antigen-presenting cells (APCs), such as macrophages (MPhi) and dendritic cells (DCs), is required for an efficient CD8 T-cell-mediated immune response. We have previously shown that co-encapsulation of antigenic protein inside pH-sensitive liposomes with listeriolysin O (LLO), a pore-forming protein of Listeria monocytogenes, generates efficient major histocompatibility complex class I (MHC I)-restricted immune responses both in vitro and in vivo. In this study, we sought to analyze the relative efficiency of LLO-mediated cytosolic delivery of liposomal antigen in two important APCs, macrophages and dendritic cells, by examining the sequential steps involved in antigen presentation to T-cells in cultured mouse bone marrow-derived MPhis (BMMPhis) and DCs (BMDCs). BMMPhis overall presented liposomal antigen better than BMDCs at a given concentration of liposomal antigen incubated with cells, and the trend was also observed after the presentation was normalized by the uptake of antigen. When soluble antigen was directly introduced into the cytosol, however, BMDCs presented the antigen more efficiently than BMMPhis. In addition, when the APCs were externally loaded with the antigenic peptide of the protein, BMDCs displayed a higher level of cell surface MHC I-peptide complexes and presented the peptide more efficiently than BMMPhis. These results combined together suggest that LLO-mediated release of liposomal antigen from the endosomal/lysosomal compartment may be more pronounced in BMMPhis than in BMDCs, and further implicates differential activity of LLO and varying efficiency of LLO-mediated endosomal escape in different antigen-presenting cell types.

Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin.

Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymatic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells. Being a hydrophilic macromolecule, however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degradation within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g. ricin). These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent. Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells. Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol. In in vitro experiments, co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of approximately 0.1 nM with an extreme efficiency requiring an incubation time of only 1 h. By contrast, cells treated with equivalent concentrations of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity. Moreover, treatment by direct intratumor injection into subcutaneous solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes containing both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates.