Dendritic Cells and Hepatocytes Use Distinct Pathways to Process Protective Antigen from Plasmodium in vivo
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Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2Kb epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.Keywords:
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Antigen cross presentation is important for effective immune responses to tumors and viral infections.Dendritic cells are professional antigen presenting cells and are unique in their ability to cross-present exogenous antigens on MHC class I molecules and activate antigen specific cytotoxic T cells.This protocol describes antigen cross presentation by dendritic cells (DCs) (bone marrow derived DCs and splenic DCs) in an in vitro and in an in vivo assay system using soluble ovalbumin protein.
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LLO is the major immuno-dominant antigen in listeriosis and is also required for protective immunity. Two forms of LLO can be observed in endosomal membranes, a LLO intact form and a Ctsd-processed LLO(1-491) form. Endosomes obtained from resting macrophages contained only LLO intact forms, while endosomes obtained from IFN-activated macrophages contained both forms. Both types of endosomes elicited LLO(90-91)/CD8(+) and LLO(189-201)/CD4(+) specific immune responses. However, only endosomes containing the Ctsd-processed LLO(1-491) form showed significant CD4(+) and CD8(+) T cell responses similar to LM infected bone marrow derived macrophages and characteristic of protective Listeria immunity. Moreover, endosomes with intact LLO could not confer protection as vaccine carriers against murine listeriosis. While endosomes with Ctsd-processed LLO(1-491) form showed a moderate ability, slightly lower than high efficiency vaccine vectors as MØ infected with LM. These studies argue that all cell-free membrane vesicles might serve as valid vaccine carriers against infectious agents. Exclusively those cell-free vesicles MIIC competent for LLO processing are protective vaccines vectors since they recruit significant numbers of mature dendritic cells to the vaccination sites and contain a LLO(1-491) form that might be accessible for MHC class I and class II antigen presentation.
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The cell biology of cross‐presentation is reviewed regarding exogenous antigen uptake, antigen degradation and entry into the major histocompatibility complex class I pathway. Whereas cross‐presentation is not associated with enhanced phagocytic ability, certain receptors may favour uptake for cross‐presentation for example mannose receptor for soluble glycoproteins. Perhaps, the defining property of the cross‐presenting cell is some specialization in host machinery for handling and transport of antigen across organelles. Both cytosolic and vacuolar pathways are discussed. Which dendritic cell (DC) subset is the cross‐presenting cell is explored. Cross‐presentation is found within the CD8 + subset resident in lymphoid organs. The role of other DC subsets (especially the migratory CD8 − DC) and the route of antigen delivery are also discussed. Further consideration is given to antigen transfer between DC subsets and differential presentation to naive vs memory T cells.
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Abstract The initiation of most cytotoxic immune responses requires MHC class I‐restricted presentation of internalized antigens to CD8 + T lymphocytes, a process called cross‐presentation. In dendritic cells (DC), the only antigen‐presenting cells that activate naive T cells, cross‐presentation is particularly efficient after internalization of opsonized antigens or immune complexes, which are cross‐presented through a proteasome‐ and transporter associated with antigen processing (TAP)‐dependent MHC class I antigen presentation pathway. We now show that FcγR‐mediated cross‐presentation is tightly regulated during DC maturation. Cross‐presentation increases soon after activation by lipopolysaccharides, and it is then inhibited in fully mature cells. The initial induction of cross‐presentation results from an increase of both antigen internalization and delivery to the cytosol, and from a slight rise in the activity of the proteasome and TAP. The subsequent block of cross‐presentation in mature DC is a consequence of the selective down‐modulation of antigen internalization and cytosolic delivery, while proteasome and TAP activities continue to rise. Therefore, FcγR‐mediated cross‐presentation is regulated during DC maturation by the selective control of antigen internalization and transport to the cytosol.
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AbstractCross-presentation of exogenous antigens by host professional antigen-presenting cells (APCs) plays a pivotal role in the initiation and development of T-cell immune responses to tumor-associated antigens, including self or mutated self-antigens derived from tumor cells, and foreign antigens derived from infectious agents. Cross-presentation requires multiple steps that involve the antigens' synthesis and compartmentalization in donor cells, packaging and delivery, and processing and presentation by MHC class I molecules on professional APCs. The intricate pathways that lead to protein degradation and the formation of MHC I-peptide complexes inside the APC are well documented for both soluble and particulate antigens. However, much less is known about how cross-presentation is regulated by the protein degradation pathways in antigen-donor cells (ADCs), including autophagy-mediated lysosomal proteolysis and proteasomal degradation. The exact nature or form of the antigens derived from donor cells at the time of delivery to the APC for cross-presentation is very controversial.This article refers to:
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The development of Ag-presenting functions by murine dendritic cells (DCs) of the CD8(+) DC lineage was studied using a Flt-3 ligand stimulated bone-marrow culture system. Although newly formed DCs of this lineage are capable of Ag uptake and efficient presentation to T cells on MHC class II, they initially lack the ability to cross-present exogenous Ags on MHC class I. Cross-presentation capacity is acquired as a subsequent maturation step, promoted by cytokines such as GM-CSF. The development of cross-presentation capacity by the DCs in these cultures may be monitored by the parallel development of DC surface expression of CD103. However, the expression of CD103 and cross-presentation capacity are not always linked; therefore, CD103 is not an essential part of the cross-presentation machinery. These results explain the considerable variability in CD103 expression by CD8(+) DCs as well as the findings that not all DCs of this lineage are capable of cross-presentation.
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Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed “cross-presentation.” The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.
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Abstract Cross-presentation allows for the presentation of exogenous viral and tumor antigens to CD8+ T cells by XCR1+ classical dendritic cells (cDC1s). Previously, the role of cross-presentation in cytotoxic T cell responses in vivo was unclear due to the lack of mouse models specifically deficient in cross-presentation. We developed a CRISPR-Cas9 screen to find novel regulators of cross-presentation and found that the protein WDFY4 is essential for cross-presentation of cell-associated antigens but not for direct presentation or presentation on MHCII. WDFY4-deficient mice had normal cDC1 development and were able to survive infections with Toxoplasma gondii through production of IL-12. However, WDFY4-deficient mice failed to prime CD8+ T cells to viral antigens and were completely unable to control immunogenic tumors. WDFY4 interacts with proteins related to vesicular formation and trafficking such as clathrin and HSP90ab1 and localizes near the cell surface, suggesting its involvement in vesicular targeting after antigen uptake during cross-presentation.
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