The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes

Abstract

Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.

A secreted pore‐forming protein modulates cellular endolysosomes to augment antigen presentation

“…Previous studies revealed that Sticholysin (St) II, an invertebrate PFP produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, was encapsulated into liposome to efficiently enhance the antigen-specific CTL responses as an adjuvant and this may not depend on its pore-forming activity. 64,65 However, βγ-CAT is a vertebrate PFP from the frog B. maxima belonging to the aerolysin family. Importantly, the enhanced antigen presentation induced by βγ-CAT depends on its capacity of pore formation in BMDCs.…”

Antigen Cross-Presentation by Macrophages

“…The cross-presenting capacities of BM macrophages seem to be higher than BM DCs when tested side-by-side using liposomes encapsulating a mixture of the model antigen ovalbumin and the pore-forming protein sticholysin II. Macrophage colony-stimulating factor–differentiated BM macrophages were better in activating the ovalbumin-recognizing B3Z CD8 + T-cell line (which does not require costimulation) than GM-CSF–differentiated BM-derived DCs in vitro ( 49 ). This could be explained by the lower ability of the DCs to internalize the antigen-containing liposomes ( 49 ).…”

“…Macrophage colony-stimulating factor–differentiated BM macrophages were better in activating the ovalbumin-recognizing B3Z CD8 + T-cell line (which does not require costimulation) than GM-CSF–differentiated BM-derived DCs in vitro ( 49 ). This could be explained by the lower ability of the DCs to internalize the antigen-containing liposomes ( 49 ). Furthermore, inhibitors of the lysosomal proteases cathepsins and leupeptin resulted in a reduced efficiency of B3Z T-cell activation by the BM macrophages, whereas a proteasome inhibitor (epoxomicin) had no effect ( 49 ).…”

Membrane remodeling by the lytic fragment of sticholysin II: implications for the toroidal pore model

“…Sticholysins I and II (StI and II) are actinoporins produced by the Caribbean Sea anemone Stichodactyla helianthus (23). These toxins are of current interest as an active component of a vaccine platform (24,25) and of immunotoxin constructs active against tumor cells (26,27). Peptides reproducing the N-terminus of StI sequence 1-31 (StI1-31) and StII sequence 1-30 (StII1-30) ( Figure 1A) can mimic the permeabilizing ability of these toxins in red blood cells and liposomes (10,12,13).…”

“…Its forms pores in human red blood cells with a similar size to those formed by the full-length toxin (9). StII1-30 has emerged as a good candidate to replace the whole toxins in some biomedical applications such as an active component of a vaccinal platform to promote the delivery of different molecules to the cytosol and enhance the immune response in anti-cancer therapies (24,25). Previously, we have analysed the conformational properties of StI1-31 and StII1-30 in solution and in membrane mimetic systems and their ability to permeabilize lipid vesicles (9,12,13,28,29).…”