Engineered Extracellular Vesicles to Enhance Antigen Presentation for Boosting Light-Driven Tumor Immunotherapy.

Extracellular vesicles (EVs) have emerged as potential tools for tumor-target therapy accompanied with activating anticancer immune responses by serving as an integrated platform, but usually suffered from the limited cross presentation of tumor-associated antigen by dendritic cells (DCs). Here, a straightforward engineering strategy to construct heat shock proteins 70 (HSP70) highly expressed EVs incapsulated with Te nanoparticles (Te@EVsHSP70 ) for tumor photothermal therapy triggering improved immunotherapy is proposed. Tumor cells are firstly used as bioreactors for intracellular synthesis of Te nanoparticles, and NIR irradiation is subsequently introduced to upregulate the expression of HSP70 to give engineered Te@EVsHSP70 through exocytosis. Te@EVsHSP70 exhibits excellent photothermal performance and enhanced tumor antigen capture capability, which induces significant immunogenic death of tumor cells and improves DCs maturation both in vitro and in vivo. Thus, the engineered EVs demonstrate superior antitumor efficacy through photothermal effect and following provoked antitumor immune responses. This work provides a facile method to fabricate multifunctional EVs-based drug delivery system for improving photothermal-triggered tumor immunotherapy.

The Role of Angiotensin-Converting Enzyme in Immunity: Shedding Light on Experimental Findings.

Angiotensin-converting enzyme (ACE) is a zinc-dependent dicarboxypeptidase with two catalytic components, which has an important role in regulating blood pressure by converting angiotensin I to angiotensin II. ACE breaks down other peptides besides angiotensin I and has a variety of physiological effects together with renal growth and reproduction in men. ACE also acts on innate and acquired immune systems by affecting macrophage and neutrophil function, and these outcomes are exacerbated due to the overexpression of ACE. Overexpression of ACE in macrophages imposes antitumor and antimicrobial response, and it enhances the ability of neutrophils to produced super peroxide that has a bactericidal effect. ACE is also known to contribute to the expression of Major Histocompatibility Complex (MHC) class I and MHC class II peptides through enzymatic alterations of these peptides. Apprehending the expression of ACE and its effects on myeloid cell (myelogenous cells) activity can be promising in therapeutic interventions, including treatment of infection and malignancy.

Antigen presentation by lung epithelial cells directs CD4+ TRM cell function and regulates barrier immunity.

Barrier tissues are populated by functionally plastic CD4+ resident memory T (TRM) cells. Whether the barrier epithelium regulates CD4+ TRM cell locations, plasticity and activities remains unclear. Here we report that lung epithelial cells, including distinct surfactant protein C (SPC)lowMHChigh epithelial cells, function as anatomically-segregated and temporally-dynamic antigen presenting cells. In vivo ablation of lung epithelial MHC-II results in altered localization of CD4+ TRM cells. Recurrent encounters with cognate antigen in the absence of epithelial MHC-II leads CD4+ TRM cells to co-express several classically antagonistic lineage-defining transcription factors, changes their cytokine profiles, and results in dysregulated barrier immunity. In addition, lung epithelial MHC-II is needed for surface expression of PD-L1, which engages its ligand PD-1 to constrain lung CD4+ TRM cell phenotypes. Thus, we establish epithelial antigen presentation as a critical regulator of CD4+ TRM cell function and identify epithelial-CD4+ TRM cell immune interactions as core elements of barrier immunity.

Exploiting B Cell Transfer for Cancer Therapy: Engineered B Cells to Eradicate Tumors.

Nowadays, cancers still represent a significant health burden, accounting for around 10 million deaths per year, due to ageing populations and inefficient treatments for some refractory cancers. Immunotherapy strategies that modulate the patient's immune system have emerged as good treatment options. Among them, the adoptive transfer of B cells selected ex vivo showed promising results, with a reduction in tumor growth in several cancer mouse models, often associated with antitumoral immune responses. Aside from the benefits of their intrinsic properties, including antigen presentation, antibody secretion, homing and long-term persistence, B cells can be modified prior to reinfusion to increase their therapeutic role. For instance, B cells have been modified mainly to boost their immuno-stimulatory activation potential by forcing the expression of costimulatory ligands using defined culture conditions or gene insertion. Moreover, tumor-specific antigen presentation by infused B cells has been increased by ex vivo antigen loading (peptides, RNA, DNA, virus) or by the sorting/ engineering of B cells with a B cell receptor specific to tumor antigens. Editing of the BCR also rewires B cell specificity toward tumor antigens, and may trigger, upon antigen recognition, the secretion of antitumor antibodies by differentiated plasma cells that can then be recognized by other immune components or cells involved in tumor clearance by antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity for example. With the expansion of gene editing methodologies, new strategies to reprogram immune cells with whole synthetic circuits are being explored: modified B cells can sense disease-specific biomarkers and, in response, trigger the expression of therapeutic molecules, such as molecules that counteract the tumoral immunosuppressive microenvironment. Such strategies remain in their infancy for implementation in B cells, but are likely to expand in the coming years.

A genotype-phenotype screening system using conditionally immortalized immature dendritic cells.

Here, we describe a protocol for CRISPR/Cas9-mediated gene knockout in conditionally immortalized immature dendritic cells (DCs), which can be limitlessly expanded before differentiation. This facilitates the genetic screening of DC functions in vitro including assessment of phagocytosis, cytokine production, expression of co-stimulatory or co-inhibitory molecules, and antigen presentation, as well as evaluation of the capacity to elicit anticancer immune responses in vivo. Altogether, these approaches described in this protocol allow investigators to link the genotype of DCs to their phenotype. For complete details on the use and execution of this protocol, please refer to Le Naour et al. (2020).

Chemotherapy agents stimulate dendritic cells against human colon cancer cells through upregulation of the transporter associated with antigen processing.

Single immunotherapy fails to demonstrate efficacy in patients with microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Research on immune reactions before and after systemic agents for mCRC is warranted. Our study examined cell line models to compare the expression of immune surface markers on colon cancer cells before and after chemotherapy agents. We also elucidated mechanisms underlying the effects of chemotherapy agents on immune surface markers. We used real-world clinical samples with NanoString analysis and the Perkin-Elmer Opal multiplex system. We established that chemotherapy agents, particularly 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, stimulated the expression of stimulatory MHC class I alleles through stimulation the pathway of transporters associated with antigen processing 1 and 2 (TAP1 and TAP2) in cell line models. Application of infected cell protein 47 (ICP-47), a specific inhibitor of the TAP1/TAP2, significantly inhibited expression of TAP1/TAP2 and also inhibited the expression of the downstream MHC class I. In the functional assay, SN-38 significantly promoted the phagocytosis of colon cancer cells by monocyte-derived dendritic cells (MoDCs). We confirmed that the expression of major histocompatibility complex (MHC) class I, significantly increased after first-line chemotherapy and targeted therapy in the samples of real-world patients with de novo mCRC. Our study provides new insights for novel immunotherapy combinations.

Oxidation inhibits autophagy protein deconjugation from phagosomes to sustain MHC class II restricted antigen presentation.

LC3-associated phagocytosis (LAP) contributes to a wide range of cellular processes and notably to immunity. The stabilization of phagosomes by the macroautophagy machinery in human macrophages can maintain antigen presentation on MHC class II molecules. However, the molecular mechanisms involved in the formation and maturation of the resulting LAPosomes are not completely understood. Here, we show that reactive oxygen species (ROS) produced by NADPH oxidase 2 (NOX2) stabilize LAPosomes by inhibiting LC3 deconjugation from the LAPosome cytosolic surface. NOX2 residing in the LAPosome membrane generates ROS to cause oxidative inactivation of the protease ATG4B, which otherwise releases LC3B from LAPosomes. An oxidation-insensitive ATG4B mutant compromises LAP and thereby impedes sustained MHC class II presentation of exogenous Candida albicans antigens. Redox regulation of ATG4B is thereby an important mechanism for maintaining LC3 decoration of LAPosomes to support antigen processing for MHC class II presentation.

The quest for faithful in vitro models of human dendritic cells types.

Dendritic cells (DCs) are mononuclear phagocytes that are specialized in the induction and functional polarization of effector lymphocytes, thus orchestrating immune defenses against infections and cancer. The population of DC encompasses distinct cell types that vary in their efficacy for complementary functions and are thus likely involved in defending the body against different threats. Plasmacytoid DCs specialize in the production of high levels of the antiviral cytokines type I interferons. Type 1 conventional DCs (cDC1s) excel in the activation of cytotoxic CD8+ T cells (CTLs) which are critical for defense against cancer and infections by intracellular pathogens. Type 2 conventional DCs (cDC2s) prime helper CD4+ T cells for the production of type 2 cytokines underpinning immune defenses against worms or of IL-17 promoting control of infections by extracellular bacteria or fungi. Hence, clinically manipulating the development and functions of DC types could have a major impact for improving treatments against many diseases. However, the rarity and fragility of human DC types is impeding advancement towards this goal. To overcome this roadblock, major efforts are ongoing to generate in vitro large numbers of distinct human DC types. We review here the current state of this research field, emphasizing recent breakthrough and proposing future priorities. We also pinpoint the necessity to develop a consensus nomenclature and rigorous methodologies to ensure proper identification and characterization of human DC types. Finally, we elaborate on how faithful in vitro models of human DC types can accelerate our understanding of the biology of these cells and the engineering of next generation vaccines or immunotherapies against viral infections or cancer.

Role for ribosome-associated quality control in sampling proteins for MHC class I-mediated antigen presentation.

Mammalian cells present a fingerprint of their proteome to the adaptive immune system through the display of endogenous peptides on MHC-I complexes. MHC-I-bound peptides originate from protein degradation by the proteasome, suggesting that stably folded, long-lived proteins could evade monitoring. Here, we investigate the role in antigen presentation of the ribosome-associated quality control (RQC) pathway for the degradation of nascent polypeptides that are encoded by defective messenger RNAs and undergo stalling at the ribosome during translation. We find that degradation of model proteins by RQC results in efficient MHC-I presentation, independent of their intrinsic folding properties. Quantitative profiling of MHC-I peptides in wild-type and RQC-deficient cells by mass spectrometry showed that RQC substantially contributes to the composition of the immunopeptidome. Our results also identify endogenous substrates of the RQC pathway in human cells and provide insight into common principles causing ribosome stalling under physiological conditions.

Myeloid Neoplasms with Elevated Plasmacytoid Dendritic Cell Differentiation Reflect the Maturation Process of Dendritic Cells.

To date, the research on dendritic cells (DCs) and their correlated neoplasms has not been clear. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) and mature plasmacytoid dendritic cell proliferation (MPDCP) are two types of malignancies originating from plasmacytoid dendritic cells (pDCs). Some evidence has indicated the existence of other pDC neoplasms. In addition, cases of myeloid neoplasms (MNs), acute myeloblastic leukemia (AML), and myelodysplastic syndrome (MDS) with increased pDCs (AML/MDS-pDCs) seem to have immature DCs according to the vaguely consistent expression of markers among MNs and pDCs, which appear to fit the developmental pattern of normal DCs. We analyzed 14 AML/MDS-pDC cases mainly for their immunophenotype by flow cytometry and inferred their CD expression pattern. The patients' clinical information and other laboratory data were collected and reviewed. AML/MDS-pDCs show a different pattern of markers from BPDCN and MPDCP. Three maturation-involved stages were found in these AML/MDS-pDCs patients. Stage I was the most immature stage and displayed an expression profile of CD34+/st+ CD117+/st+ BDCA2- BDCA4- CD123+ HLA-DR+/st+ CD4- CD45dim+ ; Stage II was the more immature stage displayed a phenotype of CD34dim+ CD117dim+ BDCA2-/dim+ BDCA4-/dim+ CD123st+ HLA-DR+/st+ CD4- CD45+ ; and Stage III was the mature stage showed CD34- CD117- BDCA2+ /BDCA4+ CD123st+ HLA-DR+/st+ CD4+ CD45+/st+ . Three maturation-involved stages overlapped well with the phenotypes of normal DC progenitors in a continuously developmental process: granulocyte, monocyte, and DC progenitors (GMDPs) and/or monocyte and DC progenitors (MDPs), common DC progenitors (CDPs), pDCs, and/or pre-DCs. In this study, we considered AML/MDS-pDCs as entities that were distinct from BPDCN and MPDCP and correlated the components of this tumor with the normal DC differentiation pathway, which provides new evidence for understanding DC neoplasms. © 2019 International Society for Advancement of Cytometry.

Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation.

The mechanisms that govern organelle adaptation and remodelling remain poorly defined. The endo-lysosomal system degrades cargo from various routes, including endocytosis, phagocytosis, and autophagy. For phagocytes, endosomes and lysosomes (endo-lysosomes) are kingpin organelles because they are essential to kill pathogens and process and present antigens. During phagocyte activation, endo-lysosomes undergo a morphological transformation, going from a collection of dozens of globular structures to a tubular network in a process that requires the phosphatidylinositol-3-kinase-AKT-mechanistic target of rapamycin (mTOR) signalling pathway. Here, we show that the endo-lysosomal system undergoes an expansion in volume and holding capacity during phagocyte activation within 2 h of lipopolysaccharides (LPS) stimulation. Endo-lysosomal expansion was paralleled by an increase in lysosomal protein levels, but this was unexpectedly largely independent of the transcription factor EB (TFEB) and transcription factor E3 (TFE3), which are known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of acute organelle expansion via mTOR Complex 1 (mTORC1)-dependent increase in translation, which appears to be mediated by both S6Ks and 4E-BPs. Moreover, we show that stimulation of RAW 264.7 macrophage cell line with LPS alters translation of a subset but not all of mRNAs encoding endo-lysosomal proteins, thereby suggesting that endo-lysosome expansion is accompanied by functional remodelling. Importantly, mTORC1-dependent increase in translation activity was necessary for efficient and rapid antigen presentation by dendritic cells. Collectively, we identified a previously unknown and functionally relevant mechanism for endo-lysosome expansion that relies on mTORC1-dependent translation to stimulate endo-lysosome biogenesis in response to an infection signal.

Cutting antigenic peptides down to size.

A critical step in antigen presentation is the degradative processing of peptides by aminopeptidases in the endoplasmic reticulum. It is unclear whether these enzymes act only on free peptides or on those bound to their major histocompatibility complex (MHC)-I-presenting molecules. A recent study examined the structure and biophysics of N-terminally extended peptides in complex with MHC-I, revealing the conformational adjustment of MHC to permit both binding of the peptide core and exposure of the peptide N terminus. These data suggest a mechanism by which aminopeptidase access is determined and offer an explanation for how longer peptides may be displayed at the cell surface.

Effects of Localization of Antigen Proteins in Antigen-Loaded Exosomes on Efficiency of Antigen Presentation.

Exosomes are considered to be vehicles of antigen delivery. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of exosomes, might affect antigen presentation after exosomes are taken up by antigen-presenting cells; however, little is known about the importance of this phenomenon. In this study, lactadherin (LA) and group-specific antigen (Gag), exosome-tropic proteins that had previously been shown to cause the localization of luciferase to the outer surface and inner surface of exosomes, respectively [ Takahashi , Y. ; J. Biotechnol. 2013 ; Charoenviriyakul , C. ; Mol. Pharm. 2018 ], were used to examine the importance of the localization of antigen proteins in antigen presentation. Human embryonic kidney cells 293 (HEK293) were selected as exosomes producing cells. First, green fluorescent protein (GFP) was used to trace intracellular behavior of antigen proteins after uptake by murine dendritic DC2.4 cells. GFP-derived fluorescence signals were detected in cells only when GFP-inner-loaded (Gag-GFP) exosomes were added to them. Then, ovalbumin (OVA) was used as a model antigen protein, and OVA-loaded exosomes were added to bone marrow-derived dendritic cells. OVA-inner-loaded (Gag-OVA) exosomes showed enhanced class I antigen presentation capacity as compared with OVA-outer-loaded (OVA-LA) exosomes. Using PKH-labeled exosomes, it was found that the localization of OVA had very little effect on the cellular uptake of exosomes. These results indicate that the loading of antigen proteins inside exosomes helps in efficient antigen presentation.

Dendritic cell activation is blunted in patients with coronary artery disease and diabetes mellitus.

BACKGROUND: It has been shown that functional status of dendritic cells (DCs) in diabetic patients with unstable angina pectoris (UAP) are more mature and activated than diabetic patients without coronary artery disease (CAD) and none diabetic patients with UAP. Accordingly we aimed to assess the activation of DCs in patients with CAD with/and without Diabetes Mellitus (DM) and compare to those in subjects with normal coronary arteries (NCA). MATERIALS AND METHODS: Twenty three patients with severe CAD who were scheduled to coronary artery by-pass grafting surgery and 6 patients with angiographycally NCAs were included in the study. Activation of peripheral blood DCs have been analyzed by flow cytometric measures of CD86 activation. RESULTS: In patients with CAD and without DM, DC activation significantly increased after stimulation of oxidesized LDL (135 ±â€¯121 vs 248 ±â€¯197 p = 0.024). However this activation didn't significantly increased in patients with CAD and DM (100 ±â€¯20 vs 120 ±â€¯97, p = 0,54). Patients with NCAs and without DM showed marked activation of CD86 after stimulation with ox-LDL. CONCLUSION: We have documented that DC activation, upon stimulation of ox-LDL has blunted in patients with CAD compared to patients with NCAs. Moreover this defective activation is more pronounced in those with diabetic patients with CAD.

Estimating the Contribution of Proteasomal Spliced Peptides to the HLA-I Ligandome.

Spliced peptides are short protein fragments spliced together in the proteasome by peptide bond formation. True estimation of the contribution of proteasome-spliced peptides (PSPs) to the global human leukocyte antigen (HLA) ligandome is critical. A recent study suggested that PSPs contribute up to 30% of the HLA ligandome. We performed a thorough reanalysis of the reported results using multiple computational tools and various validation steps and concluded that only a fraction of the proposed PSPs passes the quality filters. To better estimate the actual number of PSPs, we present an alternative workflow. We performed de novo sequencing of the HLA-peptide spectra and discarded all de novo sequences found in the UniProt database. We checked whether the remaining de novo sequences could match spliced peptides from human proteins. The spliced sequences were appended to the UniProt fasta file, which was searched by two search tools at a false discovery rate (FDR) of 1%. We find that 2-6% of the HLA ligandome could be explained as spliced protein fragments. The majority of these potential PSPs have good peptide-spectrum match properties and are predicted to bind the respective HLA molecules. However, it remains to be shown how many of these potential PSPs actually originate from proteasomal splicing events.

STIM1 promotes migration, phagosomal maturation and antigen cross-presentation in dendritic cells.

Antigen cross-presentation by dendritic cells (DC) stimulates cytotoxic T cell activation to promote immunity to intracellular pathogens, viruses and cancer. Phagocytosed antigens generate potent T cell responses, but the signalling and trafficking pathways regulating their cross-presentation are unclear. Here, we show that ablation of the store-operated-Ca2+-entry regulator STIM1 in mouse myeloid cells impairs cross-presentation and DC migration in vivo and in vitro. Stim1 ablation reduces Ca2+ signals, cross-presentation, and chemotaxis in mouse bone-marrow-derived DCs without altering cell differentiation, maturation or phagocytic capacity. Phagosomal pH homoeostasis and ROS production are unaffected by STIM1 deficiency, but phagosomal proteolysis and leucyl aminopeptidase activity, IRAP recruitment, as well as fusion of phagosomes with endosomes and lysosomes are all impaired. These data suggest that STIM1-dependent Ca2+ signalling promotes the delivery of endolysosomal enzymes to phagosomes to enable efficient cross-presentation.

NKT-cell glycolipid agonist as adjuvant in synthetic vaccine.

NKT cells are CD1d-restricted, glycolipid antigen-reactive, immunoregulatory T lymphocytes that can serve as a bridge between the innate and adaptive immunities. NKT cells have a wide range of therapeutic application in autoimmunity, transplant biology, infectious disease, cancer, and vaccinology. Rather than triggering "danger signal" and eliciting an innate immune response, αGalCer-based NKT-cell agonist act via a unique mechanism, recruiting NKT cells which play a T helper-like role even without peptide as Th epitope. Importantly, the non-polymorphism of CD1d render glycolipid a universal helper epitope, offering the potential to simplify the vaccine construct capable of eliciting consistent immune response in different individuals. This review details recent advances in the design of synthetic vaccines using NKT-cell agonist as adjuvant, highlighting the role of organic synthesis and conjugation technique to enhance the immunological actives and to simplify the vaccine constructs.

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export.

Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8+ T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and ß2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.