Classical Processing by Phagosome-to-Cytosol Transport and Cross-Presentation of the Entrapped Antigen Promotes Endocytosis and MHC Class I Recognition of Archaeosome Adjuvant Phosphatidylserine Receptor-Mediated

Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.10 (CD4 (cid:1) cells expressing OVA TCR) or OT1 ( > 90% CD8 (cid:1) OVA cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2 (cid:1) ) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1 (cid:1) recipient mice, there was profound accumulation and cycling of donor-speciﬁc cells, and differentiation of H-2K b Ova CD8 (cid:1) T cells into CD44 high CD62L low effectors. Both macrophages and dendritic cells (DCs) efﬁciently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-speciﬁc receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating speciﬁc recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidiﬁcation blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidiﬁcation-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, con-ﬁrming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inﬂammation. Phosphatidylserine-speciﬁc receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy. The Journal of Immunology, 2004, 173: 566–578. washed and ﬁxed, and used to stimulate either CD8OVA1.3 (10 6 /well) or OT1 (3 (cid:2) 10 5 /well). Proliferation of CD8OVA 1.3 was based on IL-2 production determined by a bioassay on HT2 cells. To determine OT1 stimulation, IFN- (cid:3) production was quantiﬁed. Inhibitors were used at optimized concentrations: 5 (cid:2) g/ml cytochalasin B, 100 (cid:2) M chloroquine, 10 (cid:2) M monensin, 200 (cid:2) g/ml leupeptin and antipain, 40 (cid:2) M tripeptide alde-hydes, 50 (cid:2) M lactacystin, and 10 (cid:2) M epoxomicin. O -phospho- L -serine and mannopentaose were used at 20 and 100 (cid:2) g/ml concentrations. For deter-mining activation, DCs (10 5 /ml) were incubated in vitro with Ag-free ar- chaeosomes or LPS. After 24 h, expression of CD40 was analyzed by ﬂow cytometry (EPICS XL; Beckman Coulter). At 72 h, MTT uptake and cy- tokine production were determined. Sandwich ELISA was used to assay IL-12p40 production, whereas a bioassay using Wehi 164-13 cells measured TNF. 37°C assay ﬁxed formaldehyde, ﬂow Fluorescent intensity of rhodamine observed