The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER.
The molecular mechanisms regulating noncanonical protein transport across cellular membranes are poorly understood. Crosspresentation of exogenous antigens on MHC I molecules by dendritic cells (DCs) generally requires antigen translocation from the endosomal compartment into the cytosol for proteasomal degradation. In this study, we demonstrate that such translocation is controlled by the endocytic receptor and regulated by ubiquitination. Antigens internalized by the mannose receptor (MR), an endocytic receptor that targets its ligands specifically toward cross-presentation, were translocated into the cytosol only after attachment of a lysin48-linked polyubiquitin chain to the cytosolic region of the MR. Furthermore, we identify TSG101 as a central regulator of MR ubiquitination and antigen translocation. Importantly, we demonstrate that MR polyubiquitination mediates the recruitment of p97, a member of the ER-associated degradation machinery that provides the driving force for antigen translocation, toward the endosomal membrane, proving the central role of the endocytic receptor and its ubiquitination in antigen translocation.
Purpose: The NLRP3 inflammasome activation has been proposed as a common mechanism for some adjuvants to boost the immune system, and cationic liposomes were reported to potentially activate the NLRP3 inflammasome. Herein, we questioned whether the NLRP3 inflammasome-activating cationic liposomes could promote antigen presentation and be applied as an immune adjuvant. In addition, we aimed to investigate the structure effect of lipid on triggering these immune responses. Materials and methods: A series of structurally similar lipids, consisting of arginine (Arg) head group and varied lengths of alkyl chains or spacers in between were used to prepare cationic liposomes. Lipopolysaccharide-primed human or murine macrophages or phorbol 12-myristate 13-acetate-primed THP-1 cells were treated with these liposomes, and interleukin (IL)-1β secretion was measured to quantify the NLRP3 inflammasome activation. Lysosome rupture was examined in THP-1 cells by the fluorescence loss of acridine orange, a lysosome dye. Further, chicken ovalbumin (OVA) was loaded on the liposome surface and applied to murine bone marrow-derived dendritic cells (BMDCs), which activate OT-I and OT-II lymphocytes upon major histocompatibility complex (MHC) class I- and class II-mediated antigen presentation, respectively. OT-I and OT-II cell division and IL-2 secretion were measured to evaluate the antigen presentation efficiency. The expressions of MHC molecules and co-stimulatory molecules ie, CD80, CD86, and CD40 on BMDCs were investigated by flow cytometry. Results: All the liposomes showed size distributions of 80–200 nm and zeta potentials of around 50 mV. A3C14 liposomes, consisting of Arg-C3-Glu2C14 lipids induced the most potent lysosome rupture and NLRP3 inflammasome activation. OVA-A3C14 also exhibited the most potent MHC class I- and class II-mediated antigen presentation in BMDCs without interfering MHC and co-stimulatory molecules. Conclusion: The hydrophobic moieties of arginine-based liposomes are crucial in stimulating innate immune cells. A3C14 liposomes were non-immunogenic but strongly activated innate immune cells and promoted antigen presentation, and therefore can be applied as immune adjuvants.
N-glycosylation is generally accepted to enhance the immunogenicity of antigens because of two main reasons. First, the attachment of glycans enables recognition by endocytic receptors like the mannose receptor (MR) and hence increased uptake by dendritic cells (DCs). Second, foreign glycans are postulated to be immunostimulatory and their recognition could induce DC activation. However, a direct comparison between the immunogenicity of N-glycosylated vs. de-glycosylated proteins in vivo and a direct effect of N-glycosylated antigens on the intrinsic capacity of DCs to activate T cells have not been assessed so far.To analyze whether enforced N-glycosylation is a suited strategy to enhance the immunogenicity of non-glycosylated antigens for vaccination studies, we targeted non-glycoproteins towards the MR by introduction of artificial N-glycosylation using the methylotrophic yeast Komagataella phaffii (previously termed Pichia pastoris). We could demonstrate that the introduction of a single N-X-S/T motif was sufficient for efficient MR-binding and internalization. However, addition of N-glycosylated proteins neither influenced DC maturation nor their general capacity to activate T cells, pointing out that enforced N-glycosylation does not increase the immunogenicity of the antigen per se. Additionally, increased antigen-specific cytotoxic T cell responses in vivo after injection of N-glycosylated compared to de-glycosylated proteins were observed but this effect strongly depended on the epitope tested. A beneficial effect of N-glycosylation on antibody production could not be detected, which might be due to MR-cross-linking on DCs and to concomitant differences in IL-6 production by CD4+ T cells.These observations point out that the effect of N-glycosylation on antigen immunogenicity can vary between different antigens and therefore might have important implications for the development of vaccines using K. phaffii.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.