Search

Nanophotonics has come to light for detecting biomolecules and pathogens in an expeditious, highly- sensitive, label-free, and multiplexed manner. This is notably essential in disease prevention, early detection, drug development, and treatment monitoring. Refractometric biosensors rely on optical resonators exhibiting a resonance, which undergoes a spectral shift when the local refractive index changes due to bioanalyte bindings. Current spectroscopic approaches to monitor these wavelength shifts are using bulky, expensive, and sophisticated instrumentations, which limit their widespread exploitation, especially in point-of-care settings. Alternatively, tracking intensity changes over a narrow spectral window can substitute for collecting spectrally resolved information over a broad bandwidth to monitor the resonance shifts. Intensity-based platforms can be as simple as on-chip light sources and imaging detectors, offering miniaturization, lower cost, and portability to nanophotonic biosensors. However, intensity-interrogation is prone to noise factors, making them unreliable. Therefore, there is a need for novel approaches for robust and accurate miniaturized nanobiosensors that can also have an easier path forward to commercialization.

Summary Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity., Graphical Abstract, Highlights • Macrophages produce abundant extracellular vesicles in tumors • Macrophage-derived extracellular vesicles (TAM-EVs) display unique proteomic profiles • TAM-EVs enhance thromboxane synthesis in cancer cells • TAM-EVs promote, rather than limit, T cell proliferation and activation, Cianciaruso et al. develop methods for the analysis of extracellular vesicles secreted by macrophages in mouse tumors (TAM-EVs). Proteomic and lipidomic profiling indicates that TAM-EVs carry modulators of inflammation and lipid metabolism that could influence the properties of the tumor immune microenvironment.

Type 1 diabetes (T1D) is an autoimmune disease characterized by circulating autoantibodies, lymphocytic infiltration of pancreatic islets of Langerhans, and cell-specific destruction of beta cells, leading to insulin deficiency and symptomatic hyperglycemia. Some of the major target autoantigens in T1D are intracellular proteins, such as GAD65, IA-2 and proinsulin, whose initial encounter with the immune system is poorly understood. Here we describe a method for culturing monolayers of primary pancreatic islet cells in vitro, which enables detailed observation of proteins and sub-cellular processes in primary human and rat beta cells by super-resolution and live-cell light microscopy. This technical advance is broadly applicable to obtaining novel biological insights as demonstrated by our identification of a mechanism to control proliferation in primary beta cells through growth and disassembly of primary cilia. This protocol also allowed us to identify two novel interrelated mechanisms for how beta cell proteins, such as the antigen GAD65, can become target of autoimmunity and initiate T1D. A triggering factor that these two pathways have in common is represented by endoplasmic reticulum (ER) stress, to which pancreatic islet beta cells are particularly susceptible, and can be induced by inflammatory factors such as Th1 cytokines. In the first mechanism, we show that ER stress in beta cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen presenting cells (APCs) and T cell stimulation compared to the non-palmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human beta cells in pancreatic sections from GAD65 autoantibody positive individuals, who have not yet progressed to clinical onset of T1D, and T1D patients with residual beta cell mass and ongoing T cell infiltration of islets. Thus, aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system following release from stressed and/or damaged beta cells, triggering autoimmunity. In the second mechanism, we present that, following a pathway originating from the Golgi, both rat and human pancreatic islets release the intracellular beta cell autoantigens GAD65, IA-2 and proinsulin in a subtype of extracellular vesicles called exosomes. Islet-exosomes are then taken up by and can activate APCs including dendritic cells. Accordingly, anchoring of GAD65 to exosome-mimetic liposomes strongly boosts antigen presentation and T cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Furthermore, cytokine-induced ER stress enhances exosome secretion by beta cells, induces exosomal release of the immunostimulatory chaperones calreticulin, Gp96 and ORP150 and increases exosomal stimulation of APCs. Therefore, stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in initiation of autoimmune responses in T1D.

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.