BRAF inhibitor resistance of melanoma cells triggers increased susceptibility to natural killer cell-mediated lysis.

Background: Targeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine-threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in patients with advanced melanoma with a mutation. However, acquired resistance occurs in the majority of patients, leading to relapse.

Shifting the Balance of Activating and Inhibitory Natural Killer Receptor Ligands on Melanoma Lines with Vemurafenib.

Over 60% of human melanoma tumors bear a mutation in the gene. The most frequent mutation is a substitution at codon 600 (V600E), leading to a constitutively active BRAF and overactivation of the MAPK pathway. Patients harboring mutated BRAF respond to kinase inhibitors such as vemurafenib.

Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.

Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e.

Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions.

Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles.

Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells.

Human macrophages and dendritic cells can equally present MART-1 antigen to CD8(+) T cells after phagocytosis of gamma-irradiated melanoma cells.

Dendritic cells (DC) can achieve cross-presentation of naturally-occurring tumor-associated antigens after phagocytosis and processing of dying tumor cells. They have been used in different clinical settings to vaccinate cancer patients. We have previously used gamma-irradiated MART-1 expressing melanoma cells as a source of antigens to vaccinate melanoma patients by injecting irradiated cells with BCG and GM-CSF or to load immature DC and use them as a vaccine.

Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation.

Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate.

MART-1- and gp100-expressing and -non-expressing melanoma cells are equally proliferative in tumors and clonogenic in vitro.

MART-1 and gp100 are prototypical melanoma antigen (Ag), but their clinical use as vaccines or as targets of cytotoxic lymphocytes achieved modest success. Possible explanations could be that as MART-1 and gp100 are melanocyte differentiation Ag, clonogenic Ag-non-expressing cells would be spared by immune effectors, or that clonogenic cells would be intrinsically resistant to cytotoxic lymphocytes. We therefore analyzed the proliferative status of MART-1/gp100-expressing and -non-expressing cells in biopsies, and the clonogenicity and sensitiveness to cytotoxic lymphocytes of the human cutaneous melanoma cell lines MEL-XY1 and MEL-XY3.

Exosome secretion: molecular mechanisms and roles in immune responses.

Exosomes are small membrane vesicles, secreted by most cell types from multivesicular endosomes, and thought to play important roles in intercellular communications. Initially described in 1983, as specifically secreted by reticulocytes, exosomes became of interest for immunologists in 1996, when they were proposed to play a role in antigen presentation. More recently, the finding that exosomes carry genetic materials, mRNA and miRNA, has been a major breakthrough in the field, unveiling their capacity to vehicle genetic messages.