A New Plasmacytoid Dendritic Cell-Based Vaccine in Combination with Anti-PD-1 Expands the Tumor-Specific CD8+ T Cells of Lung Cancer Patients.

The purpose of immune checkpoint inhibitor (ICI)-based therapies is to help the patient's immune system to combat tumors by restoring the immune response mediated by CD8+ cytotoxic T cells. Despite impressive clinical responses, most patients do not respond to ICIs. Therapeutic vaccines with autologous professional antigen-presenting cells, including dendritic cells, do not show yet significant clinical benefit. To improve these approaches, we have developed a new therapeutic vaccine based on an allogeneic plasmacytoid dendritic cell line (PDC*line), which efficiently activates the CD8+ T-cell response in the context of melanoma. The goal of the study is to demonstrate the potential of this platform to activate circulating tumor-specific CD8+ T cells in patients with lung cancer, specifically non-small-cell lung cancer (NSCLC). PDC*line cells loaded with peptides derived from tumor antigens are used to stimulate the peripheral blood mononuclear cells of NSCLC patients. Very interestingly, we demonstrate an efficient activation of specific T cells for at least two tumor antigens in 69% of patients irrespective of tumor antigen mRNA overexpression and NSCLC subtype. We also show, for the first time, that the antitumor CD8+ T-cell expansion is considerably improved by clinical-grade anti-PD-1 antibodies. Using PDC*line cells as an antigen presentation platform, we show that circulating antitumor CD8+ T cells from lung cancer patients can be activated, and we demonstrate the synergistic effect of anti-PD-1 on this expansion. These results are encouraging for the development of a PDC*line-based vaccine in NSCLC patients, especially in combination with ICIs.

Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons.

Amyloid beta peptide (Aß), the main component of senile plaques of Alzheimer's disease brains, is produced by sequential cleavage of amyloid precursor protein (APP) and of its C-terminal fragments (CTFs). An unanswered question is how amyloidogenic peptides spread throughout the brain during the course of the disease. Here, we show that small lipid vesicles called exosomes, secreted in the extracellular milieu by cortical neurons, carry endogenous APP and are strikingly enriched in CTF-α and the newly characterized CTF-η. Exosomes from N2a cells expressing human APP with the autosomal dominant Swedish mutation contain Aß peptides as well as CTF-α and CTF-η, while those from cells expressing the non-mutated form of APP only contain CTF-α and CTF-η. APP and CTFs are sorted into a subset of exosomes which lack the tetraspanin CD63 and specifically bind to dendrites of neurons, unlike exosomes carrying CD63 which bind to both neurons and glial cells. Thus, neuroblastoma cells secrete distinct populations of exosomes carrying different cargoes and targeting specific cell types. APP-carrying exosomes can be endocytosed by receiving cells, allowing the processing of APP acquired by exosomes to give rise to the APP intracellular domain (AICD). Thus, our results show for the first time that neuronal exosomes may indeed act as vehicles for the intercellular transport of APP and its catabolites.

Disruption of amyloid precursor protein ubiquitination selectively increases amyloid ß (Aß) 40 levels via presenilin 2-mediated cleavage.

Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid ß (Aß) peptide, derived from cleavage of amyloid precursor protein (APP) by ß- and γ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affecting APP accumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aß40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aß40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aß40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2-mediated processing of a larger pool of APP-CTF on the endosomal membrane.

Efficient inhibition of infectious prions multiplication and release by targeting the exosomal pathway.

Exosomes are secreted membrane vesicles of endosomal origin present in biological fluids. Exosomes may serve as shuttles for amyloidogenic proteins, notably infectious prions, and may participate in their spreading in vivo. To explore the significance of the exosome pathway on prion infectivity and release, we investigated the role of the endosomal sorting complex required for transport (ESCRT) machinery and the need for ceramide, both involved in exosome biogenesis. Silencing of HRS-ESCRT-0 subunit drastically impairs the formation of cellular infectious prion due to an altered trafficking of cholesterol. Depletion of Tsg101-ESCRT-I subunit or impairment of the production of ceramide significantly strongly decreases infectious prion release. Together, our data reveal that ESCRT-dependent and -independent pathways can concomitantly regulate the exosomal secretion of infectious prion, showing that both pathways operate for the exosomal trafficking of a particular cargo. These data open up a new avenue to regulate prion release and propagation.

Exosomes as a novel way of interneuronal communication.

Exosomes are small extracellular vesicles which stem from endosomes fusing with the plasma membrane; they contain lipids, proteins and RNAs that are able to modify receiving cells. Functioning of the brain relies on synapses, and certain patterns of synaptic activity can change the strength of responses at sparse groups of synapses, to modulate circuits underlying associations and memory. These local changes of the synaptic physiology in one neuron driven by another have, so far, been explained by classical signal transduction modulating transcription, translation and post-translational modifications. We have accumulated in vitro evidence that exosomes released by neurons in a way depending on synaptic activity can be recaptured by other neurons. Some lipids, proteins and RNAs contained in exosomes secreted by emitting neurons could directly modify signal transduction and protein expression in receiving cells. Exosomes may be an ideal mechanism for anterograde and retrograde information transfer across synapses underlying local changes in synaptic plasticity. Exosomes might also participate in the spreading across the nervous system of pathological proteins such as PrPSc (abnormal disease-specific conformation of prion protein), APP (amyloid precursor protein) fragments, phosphorylated tau or α-synuclein.

Leveraging a powerful allogeneic dendritic cell line towards neoantigen-based cancer vaccines.

In recent years, immunotherapy has finally found its place in the anti-cancer therapeutic arsenal, even becoming standard of care as first line treatment for metastatic forms. The clinical benefit provided by checkpoint blockers such as anti-PD-1/PD-L1 in many cancers revolutionized the field. However, too many patients remain refractory to these treatments due to weak baseline anti-cancer immunity. There is therefore a need to boost the frequency and function of patients' cytotoxic CD8+ cellular effectors by targeting immunogenic and tumor-restricted antigens, such as neoantigens using an efficient vaccination platform. Dendritic cells (DC) are the most powerful immune cell subset for triggering cellular immune response. However, autologous DC-based vaccines display several limitations, such as the lack of reproducibility and the limited number of cells that can be manufactured. Here we discuss the advantages of a new therapeutic vaccine based on an allogeneic Plasmacytoid DC cell line, which is easy to produce and represents a powerful platform for priming and expanding anti-neoantigen cytotoxic CD8+ T-cells.

Engineering a Human Plasmacytoid Dendritic Cell-Based Vaccine to Prime and Expand Multispecific Viral and Tumor Antigen-Specific T-Cells.

Because dendritic cells are crucial to prime and expand antigen-specific CD8+ T-cells, several strategies are designed to use them in therapeutic vaccines against infectious diseases or cancer. In this context, off-the-shelf allogeneic dendritic cell-based platforms are more attractive than individualized autologous vaccines tailored to each patient. In the present study, a unique dendritic cell line (PDC*line) platform of plasmacytoid origin, already used to prime and expand antitumor immunity in melanoma patients, was improved thanks to retroviral engineering. We demonstrated that the clinical-grade PDC*line, transduced with genes encoding viral or tumoral whole proteins, efficiently processed and stably presented the transduced antigens in different human leukocyte antigen (HLA) class I contexts. Moreover, the use of polyepitope constructs allowed the presentation of immunogenic peptides and the expansion of specific cytotoxic effectors. We also demonstrated that the addition of the Lysosome-associated membrane protein-1 (LAMP-1) sequence greatly improved the presentation of some peptides. Lastly, thanks to transduction of new HLA molecules, the PDC platform can benefit many patients through the easy addition of matched HLA-I molecules. The demonstration of the effective retroviral transduction of PDC*line cells strengthens and broadens the scope of the PDC*line platform, which can be used in adoptive or active immunotherapy for the treatment of infectious diseases or cancer.

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins.

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA(2)-IVA, the calcium-independent iPLA(2)-VIA, and the secreted sPLA(2)-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of phospholipase A(2) (PLA(2))and PLD(2). A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-prostaglandinJ(2) (15-d PGJ(2)), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.

Selective activation of nuclear phospholipase D-1 by g protein-coupled receptor agonists in vascular smooth muscle cells.

Recent studies highlight the existence of an autonomous nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PCs as a source of second messengers and, particularly, nuclear phospholipase D (PLD) identification in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). Using immunoblot experiment, in vitro PLD assay with fluorescent substrate and confocal microscopy analysis, we demonstrated that only PLD1 is expressed in VSMC nucleus, whereas PLD1 and PLD2 are present in VSMC. Inhibition of RhoA and protein kinase Czeta (PKCzeta) by C3-exoenzyme and PKCzeta pseudosubstrate inhibitor, respectively, conducted a decrease of phosphatidylethanol production. On the other hand, treatment of intact VSMCs, but not nuclei, with phosphoinositide 3-kinase (PI3K) inhibitors prevented partially nuclear PLD1 activity, indicating for the first time that PI3K may have a role in nuclear PLD regulation. In addition, lysophosphatidic acid (LPA) or angiotensin II treatment of VSMCs resulted in an increase of intranuclear PLD activity, whereas platelet-derived growth factor and epidermal growth factor have no significant effect. Moreover, pertussis toxin induced a decrease of LPA-stimulated nuclear PLD1 activity, suggesting that heterotrimeric G(i)/G(0) protein involvement in intranuclear PLD1 regulation. We also show that LPA-induced nuclear PLD1 activation implied PI3K/PKCzeta pathway activation and PKCzeta nuclear translocation as well as nuclear RhoA activation. Thus, the characterization of an endogenous PLD1 that could regulate PC metabolism inside VSMC nucleus provides a new role for this enzyme in control of vascular fibroproliferative disorders.

Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies.

Exosomes are part of the family of "bioactive vesicles" and appear to be involved in distal communications between cells. They vehiculate bioactive lipids and lipolytic enzymes and their biogenesis require specific lipids and a membrane reorganisation. Their biogenesis pathway could be a way to secrete enzymes involved in lipid signalling and to generate "particulate agonists". However, this pathway seems also to be used by pathogens such as HIV. This review will consider several aspects of lipidomics studies which might help to understand the fate and role of these fascinating vesicles.

Purification and Analysis of Exosomes Released by Mature Cortical Neurons Following Synaptic Activation.

Exosomes are vesicles released by most cells into their environment upon fusion of multivesicular endosomes with the plasma membrane. Exosomes are vesicles of 60-100 nm in diameter, floating in sucrose at a density of ~1.15 g/mL and carrying a number of marker proteins such as Alix, Tsg101, and Flotillin-1. We use dissociated cortical neurons cultured for around two weeks as exosome-releasing cells. In these conditions, neurons make mature synapses and form networks that can be activated by physiological stimuli. Here, we describe methods to culture differentiated cortical neurons, induce exosome release by increasing glutamatergic synapse activity, and purify exosomes by differential centrifugations followed by density separation using sucrose gradients. These protocols allow purification of neuronal exosomes released within minutes of activation of glutamatergic synapses.

Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization.

Exosomes are small vesicles secreted from multivesicular bodies, which are able to stimulate the immune system leading to tumour cell eradication. We have analysed lipids of exosomes secreted either upon stimulation from rat mast cells (RBL-2H3 cells), or constitutively from human dendritic cells. As compared with parent cells, exosomes displayed an enrichment in sphingomyelin, but not in cholesterol. Phosphatidylcholine content was decreased, but an enrichment was noted in disaturated molecular species as in phosphatidylethanolamines. Lyso(bis)phosphatidic acid was not enriched in exosomes as compared with cells. Fluorescence anisotropy demonstrated an increase in exosome-membrane rigidity from pH 5 to 7, suggesting their membrane reorganization between the acidic multivesicular body compartment and the neutral outer cell medium. NMR analysis established a bilayer organization of exosome membrane, and ESR studies using 16-doxyl stearic acid demonstrated a higher flip-flop of lipids between the two leaflets as compared with plasma membrane. In addition, the exosome membrane exhibited no asymmetrical distribution of phosphatidylethanolamines. Therefore exosome membrane displays a similar content of the major phospholipids and cholesterol, and is organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, we observed tight lipid packing at neutral pH and a rapid flip-flop between the two leaflets of exosome membranes. These parameters could be used as a hallmark of exosomes.

PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes.

Exosomes are small vesicles secreted by different immune cells and which display anti-tumoral properties. Stimulation of RBL-2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.

Exosomes secreted by cortical neurons upon glutamatergic synapse activation specifically interact with neurons.

Exosomes are nano-sized vesicles of endocytic origin released into the extracellular space upon fusion of multivesicular bodies with the plasma membrane. Exosomes represent a novel mechanism of cell-cell communication allowing direct transfer of proteins, lipids and RNAs. In the nervous system, both glial and neuronal cells secrete exosomes in a way regulated by glutamate. It has been hypothesized that exosomes can be used for interneuronal communication implying that neuronal exosomes should bind to other neurons with some kind of specificity. Here, dissociated hippocampal cells were used to compare the specificity of binding of exosomes secreted by neuroblastoma cells to that of exosomes secreted by cortical neurons. We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells. In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons. Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication.

Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.