Preparation of Poly-Lactic-Co-Glycolic Acid Nanoparticles in a Dry Powder Formulation for Pulmonary Antigen Delivery

One of the key requirements for successful vaccination via the mucosa is particulate antigen uptake. Poly-lactic-co-glycolic acid (PLGA) particles were chosen as well-known model carriers and ovalbumin (OVA) as the model antigen. Aiming at application to the respiratory tract, which allows direct interaction of the formulation with the mucosal immune system, this work focuses on the feasibility of delivering the antigen in a nanoparticulate carrier within a powder capable of pulmonary delivery. Further requirements were adequate antigen encapsulation in order to use the characteristics of the particulate carrier for (tunable) antigen release, and capability of the production process for industrialisation (realisation in industry). For an effective particulate antigen uptake, nanoparticles with a size of around 300 nm were prepared. For this, two production methods for nanoparticles, solvent change precipitation and the double emulsion method, were evaluated with respect to antigen incorporation, transfer to a dry powder formulation, redispersion and antigen release characteristics. A spray drying step was included in the production procedure in order to obtain a respirable powder with an aerodynamic particle size of between 0.5 and 5 μm. The dried products were characterised for particle size, dispersibility and aerodynamic behaviour, as well as for immune response and cytotoxicity in cell culture models. It could be shown that the double emulsion method is suitable to prepare nanoparticles (270 nm) and to incorporate the antigen. By modifying the production method to prepare porous particles, it was possible to obtain an acceptable antigen release while maintaining an antigen load of about 10%. By the choice of polyvinyl alcohol as a stabiliser, nanoparticles could be dried and redispersed without further excipients and the production steps were capable of realisation in industry. Aerodynamic characteristics were good with a mass median aerodynamic diameter of 3.3 µm upon dispersion from a capsule-based inhaler.

Gene Expression Profiling of Lymph Node Sub-Capsular Sinus Macrophages in Cancer

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We and others have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors progress faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. Imaging of mouse and patient lymph nodes confirmed the presence of the top differentially expressed genes. These results suggest that SSMs respond to tumor formation by upregulating the machinery necessary for presentation of tumor particulate antigens to B cells.

Gene expression profiling of lymph node sub-capsular sinus macrophages in cancer

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors grow faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. These results suggest that SSMs may be able to capture immune complexes for antigen processing and presentation to B and T cells on both MHC class I and II.

EBI2-mediated bridging channel positioning supports splenic dendritic cell homeostasis and particulate antigen capture

Splenic dendritic cells (DCs) present blood-borne antigens to lymphocytes to promote T cell and antibody responses. The cues involved in positioning DCs in areas of antigen exposure in the spleen are undefined. Here we show that CD4+ DCs highly express EBI2 and migrate to its oxysterol ligand, 7α,25-OHC. In mice lacking EBI2 or the enzymes needed for generating normal distributions of 7α,25-OHC, CD4+ DCs are reduced in frequency and the remaining cells fail to situate in marginal zone bridging channels. The CD4+ DC deficiency can be rescued by LTβR agonism. EBI2-mediated positioning in bridging channels promotes DC encounter with blood-borne particulate antigen. Upon exposure to antigen, CD4+ DCs move rapidly to the T-B zone interface and promote induction of helper T cell and antibody responses. These findings establish an essential role for EBI2 in CD4+ DC positioning and homeostasis and in facilitating capture and presentation of blood-borne particulate antigens.