Christian Bonnerot

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

BackgroundDC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome.Patients and methodsExosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 × 1014 molecules) or peptides (10 versus 100 μg/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression.ResultsThe GMP process allowed to harvest about 5 × 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood.ConclusionThe first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.

Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization

Exosomes are small vesicles secreted from multivesicular bodies, which are able to stimulate the immune system leading to tumour cell eradication. We have analysed lipids of exosomes secreted either upon stimulation from rat mast cells (RBL-2H3 cells), or constitutively from human dendritic cells. As compared with parent cells, exosomes displayed an enrichment in sphingomyelin, but not in cholesterol. Phosphatidylcholine content was decreased, but an enrichment was noted in disaturated molecular species as in phosphatidylethanolamines. Lyso(bis)phosphatidic acid was not enriched in exosomes as compared with cells. Fluorescence anisotropy demonstrated an increase in exosome-membrane rigidity from pH 5 to 7, suggesting their membrane reorganization between the acidic multivesicular body compartment and the neutral outer cell medium. NMR analysis established a bilayer organization of exosome membrane, and ESR studies using 16-doxyl stearic acid demonstrated a higher flip-flop of lipids between the two leaflets as compared with plasma membrane. In addition, the exosome membrane exhibited no asymmetrical distribution of phosphatidylethanolamines. Therefore exosome membrane displays a similar content of the major phospholipids and cholesterol, and is organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, we observed tight lipid packing at neutral pH and a rapid flip-flop between the two leaflets of exosome membranes. These parameters could be used as a hallmark of exosomes.

Fc receptor signaling and trafficking: a connection for antigen processing.

Summary: Most hematopoietic cells express a wide variety of receptors for tbe Fc portion of immunoglobulins (FcR) belonging to the immun‐noreceptor family. FcRs are multichain complexes composed of ligand‐binding a chains, which determine Ig binding, and signal tranduction subunits, bearing a conserved immunoreceptor tyrosine‐based activation motif (ITAM). Besides signaling, most FcyRs also efficiently internalize antigen‐antibodies complexes and thus induce efficient processing of antigens into peptides presented by major histocompatibility complex (MHC) class II molecules. Importantly, ITAMs and cyiosolic effectors of cell signaling also determine FcyRs endocytic transport and antigen presentation capacities.

PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes

Exosomes are small vesicles secreted by different immune cells and which display anti‐tumoral properties. Stimulation of RBL‐2H3 cells with ionomycin triggered phospholipase D2 (PLD2) translocation from plasma membrane to intracellular compartments and the release of exosomes. Although exosomes carry the two isoforms of PLD, PLD2 was enriched and specifically sorted on exosomes when overexpressed in cells. PLD activity present on exosomes was clearly increased following PLD2 overexpression. PLD2 activity in cells was correlated to the amount of exosome released, as measured by FACS. Therefore, the present work indicates that exosomes can vehicle signaling enzymes.

Role of B-cell and Fc receptors in the selection of T-cell epitopes

The role of specific receptors in antigen internalization and presentation to helper T lymphocytes has been known for more than ten years. However, recent work indicates that internalization may not always be sufficient for antigen presentation. Indeed, antigen receptors such as B-cell receptors and Fc receptors may also be involved in the post-endocytic transport events that determine selectively the delivery of antigens to different endocytic compartments and thereby the presentation of different T-cell epitopes.

BCR-bound antigen is targeted to exosomes in human follicular lymphoma B-cells.

Background information. Exosomes are small membrane vesicles secreted by several cell types during exocytic fusion of multivesicular bodies with the plasma membrane. Exosomes from tumour cells can transfer antigens from cell to cell, a property favouring antigen‐specific immune responses in vitro and in vivo, and are thus an interesting putative therapeutic tool in human cancers. Exosomes have been well studied in EBV (Epstein—Barr virus)‐transformed human B‐cell lines; however, biological stimuli regulating exosome secretion quantitatively and/or qualitatively still remain poorly defined.

The murine Fc-gamma (Fcγ) receptor type II B1 is a tumorigenicity-enhancing factor in polyoma-virus-transformed 3T3 cells

The murine receptor for the Fc portion of IgG is a molecule expressed by cells of the immune system. This study suggests the hypothesis that Fcγ receptor type II B1 (FcγRIIB1) functions as a progression‐enhancing factor when expressed ectopically on non‐lymphoid tumor cells. It has been shown previously that BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) do not express FcγRII but acquire the expression of this receptor following an in vivo passage in syngeneic mice. The specific FcγRII transcript present in tumor cells was identified in this report as FcγRIIB1 (B1). In order to determine whether or not the ectopically expressed FcγRII plays a role in the progression of these transformed cells, PyV‐transformed 3T3 cells were transfected with B1‐cDNA. The B1 transfected cells were tested for their ability to form local tumors in syngeneic mice, as compared to transfected cells which express the co‐transfecting neomycine resistance (neores) DNA alone or together with the lacZ gene. FcγRIIB1 expressors exhibited a significantly higher tumorigenic phenotype than FcR‐negative controls, though both types of cells exhibited the same growth curve in vitro. The ability of FcγRIIB1 to act as a potentially tumorigenicity‐enhancing factor was also demonstrated as FcγRII was expressed by tumor cells, originating from inoculated FcγRIIB1‐transfected cells, or from inoculation of a mixture of receptor‐positive and ‐negative cells. B1‐expressing cells dominated the tumor‐cell population over non‐expressors. This dominance strengthened the hypothesis that FcR plays a role in tumor progression in vivo.

Contribution of the intracellular domain of murine FC‐gamma receptor type IIB1 to its tumor‐enhancing potential

We have previously shown that Fc gamma receptor type II B1 (FcγRIIB1), when expressed on non‐lymphoid tumor cells, significantly enhanced their tumorigenic phenotype. This study elucidates the role of the intracellular domain of FcγRIIB1 in the enhancement of the malignant phenotype of polyoma‐transformed 3T3 cells. We investigated the tumorigenic potential conferred by different variants of the receptor: FcγRIIB1, a full‐length receptor (B1) whose intracellular region is encoded by exons 8, 9 and 10; FcγRIIB2, a spliced variant (B2) whose cytoplasmic domain comprises exons 9 and 10 and lacks exon 8; and FcγRIIB1‐CT53, a deleted mutant whose cytoplasmic domain contains the fragment encoded by exon 8 alone. We have investigated various properties of cells transfected with each of the above variants: tumorigenicity in syngeneic mice, formation of colonies in soft agar, growth rate, production of soluble receptor and capping of the ligand‐bound receptor. Results show that while the presence of exon 8 did not enhance growth rate in vitro or production of soluble FcγR, it did enhance the tumorigenic phenotype of transfected cells (both in vivo and in vitro growth in soft agar). B1‐expressing cells exhibited a significantly higher tumorigenic phenotype than B2 cells. The presence of exon 8 alone (CT53 mutant) conferred the transfected cells a higher tumorigenic phenotype than FcγR‐negative control cells but lower than intact B1 or B2 cells, indicating that the presence of B1‐specific exon 8 is not sufficient but that the presence of an intact B1 intracellular domain is essential, for conferring the high tumorigenicity phenotype upon cells. We conclude that the capping, following ligand binding contributed by exon 8, and the function contributed by the specific localization of exons 9 and 10 in B1 cells may determine their malignant phenotype.

Soluble Fcγ Receptors: Interaction with Ligands and Biological Consequences

Soluble Fcγ receptors are produced by cleavage of the membrane receptors or by alternative splicing. They are found in biologic fluids. After a brief description of the structure and mode of production of soluble FcγR, we address the question of ligands and function of the soluble FcγR by using recombinant molecules and transgenic animals. We show that soluble FcγR are not only IgG-binding factors which interfere with, and block, Fc-dependent immune reactions but also molecules that interact, in vitro, with non-Ig-ligands such as CR3 and CR4 and are trigger or regulate immune functions via these receptors.

Phosphoinositide 3-Kinase Activation by Igβ Controls de Novo Formation of an Antigen-processing Compartment

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igα and Igβ. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igβ but not Igα. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igβ. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igβ but not Igα induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igβ couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.