Complete regression of a previously untreated melanoma brain metastasis with ipilimumab.

Anecdotal cases of partial or complete responses have been reported after ipilimumab therapy for stage IV melanoma with brain metastasis treated earlier by surgery or radiosurgery. We report the first case of ipilimumab monotherapy resulting in durable complete remission of untreated, progressive brain metastases in a patient with stage IV melanoma. This case and earlier reports provide support for the further evaluation of ipilimumab in melanoma patients with brain metastasis.

IL-2 production by dendritic cells is not critical for the activation of cognate and innate effectors in draining lymph nodes.

Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells.

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.

Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection.

Ideal vaccines should be stable, safe, molecularly defined, and out-of-shelf reagents efficient at triggering effector and memory Ag-specific T cell-based immune responses. Dendritic cell-derived exosomes could be considered as novel peptide-based vaccines because exosomes harbor a discrete set of proteins, bear functional MHC class I and II molecules that can be loaded with synthetic peptides of choice, and are stable reagents that were safely used in pioneering phase I studies. However, we showed in part I that exosomes are efficient to promote primary MHC class I-restricted effector CD8(+) T cell responses only when transferred onto mature DC in vivo. In this work, we bring evidence that among the clinically available reagents, Toll-like receptor 3 and 9 ligands are elective adjuvants capable of triggering efficient MHC-restricted CD8(+) T cell responses when combined to exosomes. Exosome immunogenicity across species allowed to verify the efficacy of good manufactory procedures-manufactured human exosomes admixed with CpG oligonucleotides in prophylactic and therapeutic settings of melanoma in HLA-A2 transgenic mice. CpG adjuvants appear to be ideal adjuvants for exosome-based cancer vaccines.

Exosome-based immunotherapy.

Exosomes are small membrane vesicles originating from late endosomes and secreted by hematopoietic and epithelial cells in culture. Exosome proteic and lipid composition is unique and might shed some light into exosome biogenesis and function. Exosomes secreted from professional antigen-presenting cells (i.e., B lymphocytes and dendritic cells) are enriched in MHC class I and II complexes, costimulatory molecules, and hsp70-90 chaperones, and have therefore been more extensively studied for their immunomodulatory capacities in vitro and in vivo. This review will present the main biological features pertaining to tumor or DC-derived exosomes, will emphasize their immunostimulatory function, and will discuss their implementation in cancer immunotherapy.

[Exosomes and anti-tumour immunotherapy]. / Exosomes et immunothérapie antitumorale.

Exosomes are 60 to 90 nm membrane vesicles originating from late endosomes and secreted from most hematopoietic and epithelial cells in vitro. B cell derived-exosome antigenicity was first reported in 1996 in MHC class II restricted CD4+ T lymphocytes. In 1998, we reported that dendritic cell derived-exosomes are immunogenic in mice leading to tumor rejection. These findings have renewed the interest in exosomes. The current challenge consists in understanding the mechanisms and the physiological relevance of exosomes that could contribute to the design of the optimal exosome based-vaccination. Here, we will focus on the biological features pertaining to dendritic cell- and tumor cell derived-exosomes and will discuss their potential clinical implementation.

Malignant effusions and immunogenic tumour-derived exosomes.

BACKGROUND: Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supernatants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells. METHODS: We isolated exosomes by ultracentrifugation on sucrose and D(2)O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes. FINDINGS: Malignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules. INTERPRETATION: Exosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.