Multiplexable and Scalable Aqueous Synthesis Platform for Oleate-Based, Bilayer-Coated Gold Nanoparticles.

Despite gold-based nanomaterials having a unique role in nanomedicine, among other fields, synthesis limitations relating to reaction scale-up and control result in prohibitively high gold nanoparticle costs. In this work, a new preparation procedure for lipid bilayer-coated gold nanoparticles in water is presented, using sodium oleate as reductant and capping agent. The seed-free synthesis not only allows for size precision (8-30 nm) but also remarkable particle concentration (10 mm Au). These reaction efficiencies allow for multiplexing and reaction standardization in 96-well plates using conventional thermocyclers, in addition to simple particle purification via microcentrifugation. Such a multiplexing approach also enables detailed spectroscopic investigation of the nonlinear growth process and dynamic sodium oleate/oleic acid self-assembly. In addition to scalability (at gram-level), resulting gold nanoparticles are stable at physiological pH, in common cell culture media, and are autoclavable. To demonstrate the versatility and applicability of the reported method, a robust ligand exchange with thiolated polyethylene glycol analogues is also presented.

A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells.

MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B-related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.

Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.

A filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer.

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.

A short hairpin RNA-based adjuvant targeting NF-κB repressor IκBα promotes migration of dermal dendritic cells to draining lymph nodes and antitumor CTL responses induced by DNA vaccination.

DNA vaccination is an attractive approach to elicit tumor-specific cytotoxic CD8+ T lymphocytes (CTL), which can mediate protective immunity against tumors. To initiate CTL responses, antigen-encoding plasmids employed for DNA vaccination need to activate dendritic cells (DC) through the stimulation of DNA-sensing innate immune receptors that converge in the activation of the master transcription factor NF-κB. To this end, NF-κB repressor IκBα needs to be degraded, allowing NF-κB to translocate to the nucleus and transcribe proinflammatory target genes, as well as its repressor IκBα. Therefore, NF-κB activation is self-limited by de novo synthesis of IκBa, which sequesters NF-κB in the cytosol. Hence, we tested whether co-delivering a shRNA-based adjuvant able to silence IκBα expression would further promote DNA-induced NFκB activation, DC activation and tumor-protective CTL responses induced by DNA vaccination in a preclinical model. First, an IκBα-targeting shRNA plasmid (shIκBα) was shown to reduce IκBα expression and promote NFκB-driven transcription in vitro, as well as up-regulate inflammatory target genes in vivo. Then, we showed that intradermal DNA electroporation induced the migration of skin migratory dendritic cells to draining lymph nodes and maturation of dermal dendritic cells (dDC). Interestingly, shIκBα further promoted the migration of mature skin migratory dendritic cells, in particular dDC, which are specialized in antigen cross-presentation and activation of CD8+ T cells. Consistently, mice vaccinated with a plasmid encoding the melanoma-associated antigen tyrosinase-related protein 2 (TRP2) in combination with shIκBα enhanced TRP2-specific CTL responses and reduced the number of lung melanoma foci in mice challenged with intravenous injection of B16F10 cells. Moreover, therapeutic vaccination with pTRP2 and shIκBα delayed the growth of B16F10 melanoma subcutaneous tumors. Our data suggest that adjuvants promoting NF-κB activation represent an attractive strategy to boost DC activation and promote the generation of tumor-protective CTL responses elicited by DNA vaccines.

Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby Favoring the Induction of CD8+ T Cell Responses.

Dendritic cell (DC) trafficking from peripheral tissues to lymph nodes (LNs) is a key step required to initiate T cell responses against pathogens as well as tumors. In this context, cellular membrane protrusions and the actin cytoskeleton are essential to guide DC migration towards chemotactic signals. Caveolin-1 (CAV1) is a scaffolding protein that modulates signaling pathways leading to remodeling of the actin cytoskeleton and enhanced migration of cancer cells. However, whether CAV1 is relevant for DC function and specifically for DC migration to LNs is unknown. Here, we show that CAV1 expression is upregulated in DCs upon LPS- and TNF-α-induced maturation. CAV1 deficiency did not affect differentiation, maturation, or the ability of DCs to activate CD8+ T cells in vitro. However, CAV1-deficient (CAV1-/-) DCs displayed reduced in vivo trafficking to draining LNs in control and inflammatory conditions. In vitro, CAV1-/- DCs showed reduced directional migration in CCL21 gradients in transwell assays without affecting migration velocity in confined microchannels or three-dimensional collagen matrices. In addition, CAV1-/- DCs displayed reduced activation of the small GTPase Rac1, a regulator of actin cytoskeletal remodeling, and lower numbers of F-actin-forming protrusions. Furthermore, mice adoptively transferred with peptide-pulsed CAV1-/- DCs showed reduced CD8+ T cell responses and antitumor protection. Our results suggest that CAV1 promotes the activation of Rac1 and the formation of membrane protrusions that favor DC chemotactic trafficking toward LNs where they can initiate cytotoxic T cell responses.