Antigen Cross-presentation In Dendritic Cells Research Articles

The alpha-adrenergic antagonist prazosin promotes cytosolic siRNA delivery from lysosomal compartments

The widespread use of small interfering RNA (siRNA) is limited by the multiple extra- and intracellular barriers upon in vivo administration. Hence, suitable delivery systems, based on siRNA encapsulation in nanoparticles or its conjugation to targeting ligands, have been developed. Nevertheless, at the intracellular level, these state-of-the-art delivery systems still suffer from a low endosomal escape efficiency. Consequently, the bulk of the endocytosed siRNA drug rapidly accumulates in the lysosomal compartment. We recently reported that a wide variety of cationic amphiphilic drugs (CADs) can promote small nucleic acid delivery from the endolysosomal compartment into the cytosol via transient induction of lysosomal membrane permeabilization. Here, we describe the identification of alternate siRNA delivery enhancers from the NIH Clinical Compound Collection that do not have the typical physicochemical properties of CADs. Additionally, we demonstrate improved endolysosomal escape of siRNA via a cholesterol conjugate and polymeric carriers with the α1-adrenergic antagonist prazosin, which was identified as the best performing delivery enhancer from the compound screen. A more detailed assessment of the mode-of-action of prazosin suggests that a different cellular phenotype compared to typical CAD adjuvants drives cytosolic siRNA delivery. As it has been described in the literature that prazosin also induces cancer cell apoptosis and promotes antigen cross-presentation in dendritic cells, the proof-of-concept data in this work provides opportunities for the repurposing of prazosin in an anti-cancer combination strategy with siRNA.

Pathogen Recognition-Driven Dendritic Cell-Specific Gene Silencing and Editing

Dendritic cells (DCs) play an essential role in both the induction of the immune response and the maintenance of immune tolerance, with any malfunction of DCs potentially causing several diseases. While gene-based therapy for DC manipulation is a promising approach, it remains challenging due to the lack of efficient delivery systems for DC targeting. Herein, we describe a novel bacterial nanomedicine (BNM) system for pathogen recognition-mediated DCs-specific gene silencing and gene editing. BNMs contain components from bacterial outer membranes and achieve efficient DC targeting through the recognition of pathogen-associated molecular patterns by pattern recognition receptors on DCs. The targeting efficiency of BNMs is reduced in DCs lacking toll-like receptor 4, which is responsible for recognizing lipopolysaccharide, a major component of the bacterial outer membrane. As a proof-of-concept demonstration, we present gene-based therapy mediated by BNMs for enhancing antigen cross-presentation in DCs, which generates a remarkable antitumor effect.

The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex

The stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor that regulates the activity of Orai plasma membrane Ca2+ channels to mediate the store-operated Ca2+ entry pathway essential for immunity. Uncoordinated 93 homolog B1 (UNC93B1) is a multiple membrane-spanning ER protein that acts as a trafficking chaperone by guiding nucleic-acid sensing toll-like receptors to their respective endosomal signaling compartments. We previously showed that UNC93B1 interacts with STIM1 to promote antigen cross-presentation in dendritic cells, but the STIM1 binding site(s) and activation step(s) impacted by this interaction remained unknown. In this study, we show that UNC93B1 interacts with STIM1 in the ER lumen by binding to residues in close proximity to the transmembrane domain. Cysteine crosslinking in vivo showed that UNC93B1 binding promotes the zipping of transmembrane and proximal cytosolic helices within resting STIM1 dimers, priming STIM1 for translocation. In addition, we show that UNC93B1 deficiency reduces store-operated Ca2+ entry and STIM1–Orai1 interactions and targets STIM1 to lighter ER domains, whereas UNC93B1 expression accelerates the recruitment of STIM1 to cortical ER domains. We conclude that UNC93B1 therefore acts as a trafficking chaperone by maintaining the pool of resting STIM1 proteins in a state primed for activation, enabling their rapid translocation in an extended conformation to cortical ER signaling compartments.

Personalized combination nano-immunotherapy for robust induction and tumor infiltration of CD8+ T cells

Identification of tumor-specific mutations, called neoantigens, offers new exciting opportunities for personalized cancer immunotherapy. However, it remains challenging to achieve robust induction of neoantigen-specific T cells and drive their infiltration into the tumor microenvironment (TME). Here, we have developed a novel polyethyleneimine (PEI)-based personalized vaccine platform carrying neoantigen peptides and CpG adjuvants in a compact nanoparticle (NP) for their spatio-temporally concerted delivery. The NP vaccine significantly enhanced activation and antigen cross-presentation of dendritic cells, resulting in strong priming of neoantigen-specific CD8+ T cells with the frequency in the systemic circulation reaching as high as 23 ± 7% after a single subcutaneous administration. However, activated CD8+ T cells in circulation exhibited limited tumor infiltration, leading to poor anti-tumor efficacy. Notably, local administration of stimulator of interferon genes (STING) agonist promoted tumor infiltration of vaccine-primed CD8+ T cells, thereby overcoming one of the major challenges in achieving strong anti-tumor efficacy with cancer vaccination. The NP vaccination combined with STING agonist therapy eliminated tumors in murine models of MC-38 colon carcinoma and B16F10 melanoma and established long-term immunological memory. Our approach provides a novel therapeutic strategy based on combination nano-immunotherapy for personalized cancer immunotherapy.

Next-Generation Immunotherapies to Improve Anticancer Immunity

Checkpoint inhibitors are widely used immunotherapies for advanced cancer. Nonetheless, checkpoint inhibitors have a relatively low response rate, work in a limited range of cancers, and have some unignorable side effects. Checkpoint inhibitors aim to reinvigorate exhausted or suppressed T cells in the tumor microenvironment (TME). However, the TME contains various other immune cell subsets that interact to determine the fate of cytotoxic T cells. Activation of cytotoxic T cells is initiated by antigen cross-presentation of dendritic cells. Dendritic cells could also release chemokines and cytokines to recruit and foster T cells. B cells, another type of antigen-presenting cell, also foster T cells and can produce tumor-specific antibodies. Neutrophils, a granulocyte cell subset in the TME, impede the proliferation and activation of T cells. The TME also consists of cytotoxic innate natural killer cells, which kill tumor cells efficiently. Natural killer cells can eradicate major histocompatibility complex I-negative tumor cells, which escape cytotoxic T cell–mediated destruction. A thorough understanding of the immune mechanism of the TME, as reviewed here, will lead to further development of more powerful therapeutic strategies. We have also reviewed the clinical outcomes of patients treated with drugs targeting these immune cells to identify strategies for improvement and possible immunotherapy combinations.

SHP-1 Regulates Antigen Cross-Presentation and Is Exploited by Leishmania to Evade Immunity

Intracellular pathogens have evolved strategies to evade detection by cytotoxic CD8+ T lymphocytes (CTLs). Here, we ask whether Leishmania parasites trigger the SHP-1-FcRγ chain inhibitory axis to dampen antigen cross-presentation in dendritic cells expressing the C-type lectin receptor Mincle. We find increased cross-priming of CTLs in Leishmania-infected mice deficient for Mincle or with a selective loss of SHP-1 in CD11c+ cells. The latter also shows improved cross-presentation of cell-associated viral antigens. CTL activation invitro reveals increased MHC class I-peptide complex expression in Mincle- or SHP-1-deficient CD11c+ cells. Neuraminidase treatment also boosts cross-presentation, suggesting that Leishmania triggers SHP-1-associated sialic-acid-binding receptors. Mechanistically, enhanced antigen processing correlates with reduced endosomal acidification in the absence of SHP-1. Finally, we demonstrate that SHP-1 inhibition improves CD11c+ cell-based vaccination against the parasite. Thus, SHP-1-mediated impairment of cross-presentation can be exploited by pathogens to evade CTLs, and SHP-1 inhibition improves CTL responses during vaccination.

Abstract 4577: ONM-500: A STING-activating therapeutic nanovaccine platform for cancer immunotherapy

Abstract Background: The efficacy of cancer vaccines requires induction of cancer-specific cytotoxic T-lymphocytes (CTL) to effectively clear established tumors. Orchestration of antigen presentation, co-stimulatory signaling, and innate cytokine signals are necessary steps for tumor-specific T cell activation. The ONM-500 nanovaccine platform utilizes a novel pH-sensitive polymer that forms an antigen-encapsulating nanoparticle and functions both as a carrier for antigen delivery to dendritic cells and as an adjuvant, activating the stimulator of interferon genes (STING) pathway and generating a CD8+ CTL response. Peptide antigens have translational challenges due to limited stability, HLA-type-specific antigen sequence recognition and presentation. Full-length protein antigens alleviate HLA subtype limitation, allowing coverage of multi-immunogenic T cell epitopes in patients. Using the ONM-500 platform with the full-length recombinant E6/E7 oncoproteins from human papillomavirus (HPV) cancers, we have demonstrated great antitumor efficacy in an HPV positive cancer model in animals. Methods: Based on the previously demonstrated STING-dependent T cell activation by ONM-500, the nanovaccine was formulated with full-length HPV16 E6/E7 proteins, and the nanoparticle properties and antigen loading were characterized. In vivo lymph node accumulation following subcutaneous administration was evaluated using fluorophore-labeled ONM-500. Direct binding of ONM-500 polymer to human STING was evaluated using a pull-down assay. Antitumor efficacy of ONM-500 nanovaccine with full length E6/E7 was evaluated using a TC-1 model which overexpresses HPV16 E6/E7 and compared with ONM-500 formulated with peptide antigen. Long-term anti-tumor memory was evaluated in a rechallenge study after 60 days in tumor-free animals. Results: Characterization of ONM-500 nanovaccine shows reproducible particle chemical and physical properties and antigen loading. The nanoparticle size substantiates the effective lymph node accumulation for antigen cross-presentation in dendritic cells following subcutaneous administration. The pull-down assay showed effective binding of the ONM-500 polymer to STING. In TC-1 tumors, ONM-500 nanovaccine containing full-length E6/E7 protein showed 100% overall survival at 55 days. Tumor growth inhibition was also improved over peptide antigen formulated ONM-500. A rechallenge study demonstrated long-term antigen-specific anti-tumor memory response. Conclusions: ONM-500 STING-activating nanovaccines effectively deliver antigens in vivo to lymph nodes to elicit an antigen-specific CTL response. The anti-tumor efficacy in the animal tumor model demonstrates the potential of ONM-500 as a general STING-activating cancer vaccine platform, and full-length E6/E7 incorporated ONM-500 is being developed for HPV-associated cancers. Citation Format: Jason Miller, Min Luo, Hua Wang, Zhaohui Wang, Xinliang Ding, Ashley Campbell, Jonathan Almazan, Stephen Gutowski, Zhijian Chen, Jinming Gao, Tian Zhao. ONM-500: A STING-activating therapeutic nanovaccine platform for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4577.

Nanoenabled Disruption of Multiple Barriers in Antigen Cross-Presentation of Dendritic Cells via Calcium Interference for Enhanced Chemo-Immunotherapy

Chemo-immunotherapy holds the advantage of specific antitumor effects by activating T cell immune response. However, the efficiency of chemo-immunotherapy is restricted to the insufficient antigen presentation of dendritic cells (DCs) in the tumor immunosuppression microenvironment. Here, we rationally designed a simple yet versatile calcium ion nanogenerator to disrupt the autophagy inhibition condition within DCs, enrich damage-associated molecular patterns (DAMPs), and attenuate acidity in the tumor microenvironment. After chemotherapy, honeycomb calcium carbonate (CaCO3) nanoparticles (OVA@CaCO3, denoted as HOCN, ovalbumin (OVA) acted as skeleton) could preferentially accumulate in the tumor and display a series of benefits for disrupting multiple barriers in antigen cross-presentation of DCs: (i) recovering cell viability of DCs by HOCN-induced tumor acidity attenuating; (ii) disrupting the autophagy inhibition condition in DCs by generating Ca2+ in cells; (iii) improving maturation of DCs by Ca2+ overloading-mediated enhanced DAMP release from tumor cells. In addition, HOCN can also disrupt the immunosuppressive microenvironment by reducing the infiltration of immunosuppressive cells and factors. We believe regulation of the intratumoral Ca2+ offers an alternative strategy for improving cancer chemo-immunotherapy.

Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells

Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1’s role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.

Delivered antigen peptides to resident CD8α+ DCs in lymph node by micelle-based vaccine augment antigen-specific CD8+ effector T cell response

Although nanoparticle vaccine is one of the promising therapeutic vaccines against cancers and many chronic infections, induction of strong and long-lasting antigen specific T cell response has still remained many challenges. A major challenge in achieving a robust CD8+ T cell response is the requirement of spatio-temporal orchestration of antigen cross-presentation in dendritic cells with innate stimulation. CD8α+ DCs are specialized for cross presentation and critical for cytotoxic T cell responses, which locate in the deeper paracortex of lymph nodes (LNs) in mice. However, due to size exclusion of compartmentalized network in LNs, nanoparticles with a radius of larger than 5 nm are difficult to access to the CD8α+ DCs. Here, we showed that polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles had an extensive contact with the resident CD8α+ DCs in LNs and delivered more OVA peptides than their free form to these DCs. Meanwhile, successfully delivering antigens into the CD8α+ DCs resulted in the increased cross presentation of antigens and the enhanced generation of effector CD8+ T cell. Our findings further demonstrated the critical role of CD8α+ DCs in cytotoxic T cell immunity in response to PEG-PE micelle-based vaccine, and also provided a valuable approach to generate T cell-mediated immune response.

Measuring the molecular mechanisms of tumor antigen cross-presentation in dendritic cells and macrophages

Abstract Objective Antigen cross-presentation is essential for initiation of an adaptive immune response against cancer and pathogens. During this process, dendritic cells (DCs) present peptides derived from ingested antigens in their major histocompatibility complex class I protein (MHC-I) to cytotoxic CD8+T cells. As a result, these CD8+T cells become activated, enabling them to eliminate infected or malignant cells in the human body. Aim of this study is to develop a quantitative method to measure cross-presentation of NY-ESO1, a tumor-specific antigen expressed by several cancer types. Methods DCs were activated and loaded with NY-ESO1-based antigen. To measure cross-priming, DCs were co-cultured with primary naive CD8+T cells. These T cells were transfected with mRNA coding for a specific T cell receptor recognizing an NY-ESO-1-derived epitope. After incubation, the activation of the cytotoxic T cells was measured with ELISA and flow cytometry. In addition, we followed the dynamics of bio-orthogonally tagged NY-ESO1 peptides over time for direct visualization of antigen cross-presentation in dendritic cells and Peripheral Blood Mononuclear Cells (PBMC’s). Results Cross-presentation of bio-orthogonal tagged NY-ESO1 could be followed over time in dendritic cells and PBMC's. Interestingly, in our PBMC screen, not only CD1c dendritic cells but also macrophages are highly able to cross-present ingested peptides. Conclusion This novel method allows to measure presentation of tumor antigens on MHC-I in DCs and primary cells of human blood. Since cross-presentation is crucial for elimination of virus-infected and malignant cells by the immune system, this knowledge could contribute to improvement of DC-based immune therapies.

Adjuvants Enhancing Cross-Presentation by Dendritic Cells: The Key to More Effective Vaccines?

Over the last decades, vaccine development has advanced significantly in pursuing higher safety with less side effects. However, this is often accompanied by a reduction in vaccine immunogenicity and an increased dependency on adjuvants to enhance vaccine potency. Especially for diseases like cancer, it is important that therapeutic vaccines contain adjuvants that promote strong T cell responses. An important mode of action for such adjuvants is to prolong antigen exposure to dendritic cells (DCs) and to induce their maturation. These mature DCs are extremely effective in the activation of antigen-specific T cells, which is a pre-requisite for induction of potent and long-lasting cellular immunity. For the activation of CD8+ cytotoxic T cell responses, however, the exogenous vaccine antigens need to gain access to the endogenous MHCI presentation pathway of DCs, a process referred to as antigen cross-presentation. In this review, we will focus on recent insights in clinically relevant vaccine adjuvants that impact DC cross-presentation efficiency, including aluminum-based nanoparticles, saponin-based adjuvants, and Toll-like receptor ligands. Furthermore, we will discuss the importance of adjuvant combinations and highlight new developments in cancer vaccines. Understanding the mode of action of adjuvants in general and on antigen cross-presentation in DCs in particular will be important for the design of novel adjuvants as part of vaccines able to induce strong cellular immunity.

Polyethyleneimine modification of aluminum hydroxide nanoparticle enhances antigen transportation and cross-presentation of dendritic cells.

BackgroundThe aim of this study was to explore the feasibility of delivering tumor antigens and enhancing the antigen cross-presentation of dendritic cells (DCs) by aluminum hydroxide nanoparticle with polyethyleneimine (PEI) modification (LV@HPA/PEI).Materials and methodsThe LV@HPA nanoparticles were modified by PEI first, then the influence of LV@HPA/PEI on DCs was examined. The distinct expression of ovalbumin (OVA) protein transported into DCs by LV@HPA/PEI was observed by flow cytometry and Western blot. The biocompatibility of LV@HPA/PEI, maturity and antigen cross-presentation of DCs was observed in vitro. Tumor derived autophagosomes (DRibbles) combined with LV@HPA/PEI were loaded into DCs, and DC vaccines were used to immunize mice. The percentage of CD3+CD8+IFN-γ+ T cells in immunized mice was determined by flow cytometry. Additionally, the functional properties of the LV@HPA/PEI-DRibble-DCs vaccine were examined in vivo in PancO2 tumor-bearing mice.ResultsIn our study, we described how LV@HPA/PEI can be a functionalized antigen delivery system with notable antigen transport effect and negligible cytotoxicity. It was found that LV@HPA/PEI could be easily internalized into DCs to assist antigen release into the cytoplasm. In addition, DCs matured gradually after loading with LV@HPA/PEI-OVA, which increased significantly the cytokine IL-12 secretion and expression of surface molecules CD80 and CD86. Interestingly, DCs loaded with LV@HPA/PEI-DRibbles could promote the activation of tumor-specific T cells both in murine and in human T cells. In the following in vivo experiments, the vaccine of LV@HPA/PEI-DRibble-DCs significantly inhibited tumor growth and improved the survival rate of the PancO2 tumor-bearing mice.ConclusionWe established a high-performance anti-tumor vaccine of DCs loaded with LV@ HPA/PEI nanoparticles and tumor-associated antigens in autophagosomes (DRibbles), which could serve as a therapeutic strategy in cancer immunotherapy.

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.

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

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 β2 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.

Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens.

Cationic liposomes (CLs) have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs), and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation – the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8+ T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I). However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8+ T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs), antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8+ T-cells. To achieve this, we have used 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8+ T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine components and subsequent immunological responses can be controlled by rational design of nanomaterials.

Disruption of key GTPase regulators of endocytic recycling compartment does not interfere with soluble antigen crosspresentation in dendritic cells.

Crosspresentation is of vital significance to immune surveillance. External antigens, soluble or solid, enter cells via endophagocytic vesicles and are eventually delivered to MHC class I molecules on the cell surface. After antigen uptake, there are three proposed pathways leading to surface MHC class I/peptide complex presentation. Direct translocation into the cytosol to use the conventional proteasome/TAP-dependent ER peptide loading; phagosome autonomous presentation following fusion with selected ER components; and endocytic recycling whereby antigens are processed within the endosome and form a complex with internalized MHC class I, for redelivery back to cell surface.1 Endocytic recycling compartment (ERC), a major perinuclear tubular network considered critical for slow endosomal recycling, has been suggested to be one route for crosspresentation.2 This pathway is particularly relevant for soluble antigen presentation, as the former two routes are mostly used to describe particulate antigen processing, involving phagosomes. Here we report the unexpected finding that disruption of key regulatory factors of ERC, small GTPase proteins ARF6, Rab11a, and Rab22a, had no effect on soluble ovalbumin (OVA) crosspresentation in a model dendritic cell system.

Molecular Mechanisms of TLR2-Mediated Antigen Cross-Presentation in Dendritic Cells

Cross-presentation is a key function of dendritic cells (DCs), which present exogenous Ags on MHC class I molecules to prime CTL responses. The effects of TLR triggering on the cross-presentation of exogenous Ags by DCs remain unclear. In this study, we used synthetic dipalmitoylated peptides and TLR2 agonist-conjugated peptides as models to elucidate the mechanisms of TLR2-mediated cross-presentation. We observed that the internalization of dipalmitoylated peptides by bone marrow-derived DCs was facilitated by TLR2 via clathrin-mediated endocytosis. The administration of these dipalmitoylated peptide-pulsed bone marrow-derived DCs eliminated established tumors through TLR2 signaling. We further demonstrated that the induction of Ag-specific CTL responses and tumor regression by dipalmitoylated peptides was TAP independent. In addition, presentation of dipalmitoylated peptides by MHC class I molecules was blocked in the presence of an endosomal acidification inhibitor (chloroquine) or a lysosomal degradation inhibitor (Z-FL-COCHO). The endocytosed dipalmitoylated peptide also passed rapidly from early endosome Ag-1-positive endosomes to RAS-related GTP-binding protein 7 (Rab7)-associated late endosomes compared with their nonlipidated counterparts. Furthermore, we found that dipalmitoylated peptide-upregulated Rab7 expression correlated with Ag presentation via the TLR2/MyD88 pathway. Both JNK and ERK signaling pathways are required for upregulation of Rab7. In summary, our data suggest that TLR2-mediated cross-presentation occurs through the upregulation of Rab7 and a TAP-independent pathway that prime CTL responses.

Reactive Oxygen Species Production in the Phagosome: Impact on Antigen Presentation in Dendritic Cells

The NADPH oxidase 2 (NOX2) is known to play a major role in innate immunity for several decades. Phagocytic cells provide host defense by ingesting microbes and destroy them by different mechanisms, including the generation of reactive oxygen species (ROS) by NOX2, a process known as oxidative burst. The phagocytic pathway of dendritic cells (DCs), highly adapted to antigen processing, has been shown to display remarkable differences compared to other phagocytes. Contrary to macrophages and neutrophils, the main function of DC phagosomes is antigen presentation rather than pathogen killing or clearance of cell debris. In the last few years, it became clear that NOX2 is also involved in the establishment of adaptive immunity. Several studies support the idea of a relationship between antigen presentation and the level of antigen degradation, the latter one being regulated by the pH and ROS within phagosomes. The regulation of phagosomal pH exerted by NOX2, and thereby of the efficacy of antigen cross-presentation in DCs, represents a clear illustration of how NOX2 can influence CD8(+) T lymphocyte responses. In this review, we want to put emphasis on the relationship between ROS generation and antigen processing and presentation, since there is growing evidence that the low levels of ROS generated by DCs play an important role in these processes. In the next years, it will be interesting to unravel possible mechanisms involved and to find other possible connections between NOX family members and adaptive immune responses.