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Originally published online as doi:10.1189/jlb.0206094 on June 29, 2006

Published online before print June 29, 2006
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(Journal of Leukocyte Biology. 2006;80:471-478.)
© 2006 by Society for Leukocyte Biology

Dendritic cell derived-exosomes: biology and clinical implementations

Nathalie Chaput*,1, Caroline Flament*, Sophie Viaud*, Julien Taieb*, Stephan Roux*, Alain Spatz{dagger}, Fabrice André*, Jean-Bernard LePecq{ddagger}, Muriel Boussac§, Jérôme Garin§, Sebastian Amigorena, Clotilde Théry and Laurence Zitvogel*

* ERM0208 Institut National de la Santé Et de la Recherche Médicale, Faculté de Médecine Paris Sud-Université Paris XI, and
{dagger} Department of Biology and Pathology, Institut Gustave Roussy, Villejuif, France;
{ddagger} Anosys Inc., Menlo Park, California;
§ Laboratoire de chimie des protéines, Centre d’Energie Atomique, Grenoble, France; and
U520/U365, INSERM, Institut Curie, Paris, France

1 Correspondence at current address: CIC Biotherapie, Institut Gustave Roussy, Villeneuf 94805, France. E-mail: chaput{at}igr.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 FORMATION AND COMPOSITION OF...
 BIOACTIVITY OF Dex: PRECLINICAL...
 RESULTS OF THE TWO...
 CONCLUSIONS AND FUTURE PROSPECTS
 REFERENCES
 
Exosomes are nanometer-sized membrane vesicles invaginating from multivesicular bodies and secreted from different cell types. They represent an "in vitro" discovery, but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition is unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation, or "Trojan horses" for viruses or prions. The potential of dendritic cell-derived exosomes (Dex) as cell-free cancer vaccines is addressed in this review. Lessons learned from the pioneering clinical trials allowed reassessment of the priming capacities of Dex in preclinical models, optimizing clinical protocols, and delineating novel, biological features of Dex in cancer patients.

Key Words: cytotoxic T lymphocytes • NK cells • tumor immunotherapy • clinical trial


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
FORMATION AND COMPOSITION OF...
 BIOACTIVITY OF Dex: PRECLINICAL...
 RESULTS OF THE TWO...
 CONCLUSIONS AND FUTURE PROSPECTS
 REFERENCES
 
Originally, the concept of the exosome as a nonplasma membrane vesicle emerged with the description of the shedding of transferring receptors during the maturation of reticulocytes into red blood cells. The release process appears to require a specific sorting into "internal vesicles" contained inside the endosomes [multivesicular bodies (MVB)], followed by the fusion of the limiting membrane of the endosomes with the plasma membrane, resulting in exosome release in the extracellular milieu [1 2 3 ]. Therefore, the "intraluminal vesicles" become "exosomes." In reticulocytes lacking lysosomes, the obsolete proteins that do not follow an intracellular degradation pathway could be eliminated following fusion of MVB and their content with cell surface [4 5 6 7 ].

Over the past decade, the potential sources and functions of exosomes have expanded significantly. Exosomes were shown to be secreted from hematopoietic {B lymphocytes, dendritic cells (DC), mast cells, T cells, platelets [8 9 10 ]}, epithelial cells (intestinal epithelial cells, tumor cells) [11 12 13 14 15 16 17 ], and others (melanoma, mesothelioma cells), which bear lysosomes, i.e., do not require exosome release for degradation of obsolete proteins. Common characteristics of exosomes include structure (vesicles limited by bilipidic layer), size (50–100 nm diameter), density (floatation at 1.13–1.21 g/ml on sucrose gradient), and overall protein composition (restricted set of cellular proteins) of cytosolic or membrane localization, generally associated to the endocytic pathway [3 ]. Exosomes from different cellular origins also bear distinct proteins of the producing cell type [1 2 3 ].

Exosomes secreted from antigen-presenting cells (APC) appear to be functionally relevant. Indeed, Raposo and colleagues [10 ] reported that B cell-derived exosomes can directly activate CD4+ T cells. Later on, exosomes secreted from mouse DC were shown to induce antitumor effects in a T cell-dependent manner [9 ]. The mechanisms by which exosomes operate their immunomodulatory capacities are detailed below.

The definition of DC-derived exosome (Dex) composition, bioactivity and pharmacoimmunology, and good manufacturing processes (GMPs) for their purification as therapeutic products has paved the way to their clinical development, as reviewed recently [18 ].


   FORMATION AND COMPOSITION OF Dex
TOP
 ABSTRACT
 INTRODUCTION
 FORMATION AND COMPOSITION OF...
BIOACTIVITY OF Dex: PRECLINICAL...
 RESULTS OF THE TWO...
 CONCLUSIONS AND FUTURE PROSPECTS
 REFERENCES
 
The characterization of exosomes has been facilitated by the process of their purification from culture supernatants, including differential ultracentrifugation and sucrose density flotation gradients [10 , 19 ]. Analyses of the purified exosome preparations have been performed in immunoelectronmicroscopy, Western blotting, mass spectrometry (proteomics), and thin-layer chromatography (lipid composition).

The protein composition of exosomes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining differs from that of whole cell lysates. Moreover, exosomes are distinct from microvesicles produced by apoptotic cells and are only secreted by living cells [20 , 21 ]. All the data obtained for exosomes derived from murine DC, human intestinal epithelial cells, and Epstein-Barr virus-transformed B lymphocytes revealed common and cell-specific constituents. Such exosomes highly enriched in major histocompatibility complex (MHC) Class II molecules, contained MHC Class I and CD1 molecules [19 , 22 ], cytosolic chaperon proteins (heat shock), subunits of trimeric G proteins, annexins and rab proteins, integrins, adhesion molecules, enzymes and elongation factors, cytoskeleton proteins, raft-associated proteins, and glycolipids. The most abundant protein family found in exosomes is tetraspanins, which are membrane proteins with four transmembrane domains involved in many different biological functions such as fusion, motility, immune stimulation, adhesion, and protein sorting [23 ]. Several members of this family, including CD9, CD37, CD53, CD63, CD81, and CD82, are highly enriched in exosomes from virtually any cell type. In Table 1 , we show the proteins found in human Dex [obtained from a GMP culture of monocyte derived-DC (MD-DC)] using time-of-flight (TOF) mass spectrometry, 80% of which are shared by mouse Dex. The specific roles of such proteins in exosome formation or function have not yet been assigned. For example, the glycosylphosphatidylinositol-anchored proteins CD55 and CD59 are associated with exosomes, protecting them from complement attack [24 ]. Another example has been the identification in exosomes of proteins that are part of the machinery involved in the biogenesis of MVB. Dex contain Tsg101 and Alix, which are components of the conserved machinery that selects ubiquinated cargo proteins for sorting to intraluminal vesicles [25 ]. Moreover, we have addressed the relevance of the most abundant proteins found on Dex such as MHC Classes I and II, CD86 molecules, intercellular adhesion molecule 1 (ICAM-1)/CD54, and a milk fat globule protein MFG-E8/lactadherin (which can interact with {alpha}Vß3 and {alpha}Vß5 expressed on human APC) for eliciting antigen-specific T cell immune responses (cf below). As we will elude, exosomal lactadherin harbored on mouse Dex is not critical for the priming of CD4+ T cell responses in mice [26 , 27 ]. However, we could not show lactadherin on human Dex prepared in GMP conditions but did observe lactadherin on ascitis fluid-derived tumor exosomes (our unpublished data). It is interesting that lactadherin can act in conjunction with vascular endothelial growth factor to promote angiogenesis [28 ]. Whether exosomal lactadherin could play a role in tumor neovascularization remains to be established.


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  		Table 1. Proteins Found in Human Dex (Obtained from a GMP Culture of MD-DC) Using Matrix-Assisted Laser Desorption/Ionization-TOF

 
Phospholipid metabolism is critical for the biogenesis of MVB. The ability of tetraspanins to partition into lipid microdomains may allow them to sort to the intralumenal vesicles. B cell-derived exosomes are enriched in sphingomyelin, cholesterol, and GM3, lipids that are important for the maintenance of rafts. Consistent with the raft-like properties of these lipids, raft proteins (flotilin, stomatin, lyn) are present in reticulocyte and B lymphocyte exosomes. Lipid analysis of mast cell and DC exosomes suggests that a hallmark of exosomes, as compared with plasma membrane, is an increase in transbilayer movements of phospholipids ("flip-flop"), leading to a loss of asymmetry of lipids in exosomal membranes. The high level of flip-flop together with their observed rigidity at neutral pH could have consequences for the ability of exosomes to fuse with other membranes and their stability in circulation [29 ]. Indeed, mast and Dex have a specific phospholipids composition different from the parent cells. The proportion of sphingomyelin was twice as high in exosomes as in the corresponding cells, whereas phosphatidylcholine was much lower in exosomes than in cells. In addition, the molar ratio diglicerides:phospholipids is decreased by 50% in exosomes. Moreover, Laulagnier et al. [29 30 31 ] have shown that phospholipase D2 is enriched in exosomes and suggested that phospholipid D activity carried by exosomes could be involved in putative signaling properties of exosomes through a second messenger such as phosphatidic acid or in interaction mechanisms between exosomes and target cells through the fusogenic properties of phosphatidic acid.


   BIOACTIVITY OF Dex: PRECLINICAL DATA
TOP
 ABSTRACT
 INTRODUCTION
 FORMATION AND COMPOSITION OF...
 BIOACTIVITY OF Dex: PRECLINICAL...
RESULTS OF THE TWO...
 CONCLUSIONS AND FUTURE PROSPECTS
 REFERENCES
 
Induction of specific T cell responses with Dex
Consistent with the disappearance of MVB in mature DC (mDC), the production of Dex is down-regulated upon maturation, suggesting that exosomes may be produced by immature DC (imDC) in peripheral tissues [20 ], and Dex from imDC (imDex) or mDC (mDex) could exhibit distinct bioactivities in line with their differential protein content. As mDC produce an average of two- to threefold fewer Dex proteins than imDC (0.2±0.1 µg/million mDC vs. 0.5±0.1 µg/million imDC [26 , 27 ]), and qualitative differences between imDex and mDex were considered irrelevant at first, pionnering studies were performed on imDex from human or mouse origin.

In the seminal work reported in 1998, Zitvogel et al. [9 ] showed that bone marrow-derived mouse DC (BM-DC) pulsed with acid-eluted tumor peptides could secrete immunogenic Dex, mediating tumor growth retardation or rejection in prophylaxis and therapeutic murine models. Cytotoxic T lymphocyte (CTL) responses recognizing syngeneic tumors could be detected in tumor-free animals and were found only in mice vaccinated with Dex harboring the appropriate MHC Class I molecules and tumor peptides [9 ]. In the next step, Lamparski and colleagues [19 ] optimized the protocol for peptide loading onto imDex in vitro. A GMP process for peptide loading onto human Dex was set up to pulse imDC with MHC Class II peptides but to directly pulse Dex with synthetic MHC Class I peptides after acid elution of the endogenous MHC Class I molecules [19 , 32 ]. This method was implemented to mouse experimental settings and allowed to demonstrate that exosomal MHC Class I and II/peptide complexes could mediate effective T cell priming and peptide-specific tumor recognition in human cultures ex vivo and tumor rejection in mice [33 34 35 ].

Using the humal leukocyte antigen (HLA)-A2/Mart1 model system, the first observation was that exosomal MHC Class I molecules loaded with tumor peptides could not efficiently prime directly naïve human CD8+ T cells or peptide-specific, MHC Class I-restricted human CTL clones [34 ]. Similar findings were found using the mouse Marilyn T cell receptor (TCR) transgenic CD4+ T cells reactive to I-Ab/H-Y complexes [33 ]. Rather, exosomal I-Ab molecules required transfer onto APC, mostly DC. Indeed, in the presence of DC but not B cells or macrophages, Dex I-Ab/H-Y could elicit the proliferation of specific Marilyn TCR transgenic CD4+ T [33 ] in vitro. In line with this result, human Dex A2/Mart1 could promote interferon-{gamma} (IFN-{gamma}) production by specific CTL when loaded onto DC but not fibroblasts or macrophages [34 ]. Similar results were obtained using naïve CD8+ T cells from melanoma patients primed ex vivo with autologous MD-DC loaded with autologous Dex A2/Mart1 [34 ]. We could show that the exosomal MHC and not the recipient DC molecules were indispensable for CD4+ or CD8+ T cell activation in vitro and in vivo [33 , 34 ]. Using the HHD2 HLA-A2 transgenic mouse model, we demonstrated that as few as 1010 HLA-A2 molecules can be transferred to 3 x 105 H-2b allogeneic BM-DC to trigger the elective expansion of A2/Mart1 tetramer binding CD8+ T cells [34 ]. An optimal response was achieved using 1011 exosomal A2 molecules. It is noteworthy that in this model system, the plateau for specific CTL expansion in the draining lymph node is reached using 3 x 105 autologous, mBM-DC pulsed with 1 uM Mart1 peptide, i.e., ~9 x 1010 surface expression of HLA-A2 molecules. Therefore, the presumption is that at the plateau phase, Dex and mDC are equally able to trigger T cell effectors 5 days after immunization in the lymph nodes.

The second observation was that the recipient DC becomes a better exosome-presenting cell when activated with lipopolysaccharide (LPS) in vitro [35 ]. As a consequence, we found that mouse Dex A2/Mart1 could only promote the differentiation of CD8+ Tc1 cells in the draining lymph nodes when combined with Toll-like receptor 3 (TLR3) or -9 agonists. Indeed, in the absence of adjuvants or mBM-DC, inoculation of DexA2/Mart1 in the footpad could not elicit the proliferation and differentiation of Tc1 lymphocytes [35 ]. Therefore, the design of an efficient imDex-based cancer vaccine best required an adjuvant that optimally mimicks or substitutes for mDC in vivo. Ligands for TLR3 or -9, such as oligodeoxynucleotide CpG oligomeric sequences or double-stranded RNA, were efficient for T cell priming and tumor retardation in mice. Dex admixed with CpG exhibited comparable efficacy as mDC to initiate peptide-specific CD8+ T cell responses and tumor rejection in mice. We established that in the transgenic HHD2 mice, bearing the melanoma B16F10, coexpressing human HLA-A2.1 and gp100 tumor antigen, 1010 exosomal MHC Class I/gp100 complexes admixed with CpG-mediated tumor rejection as efficiently as 3 x 105 mDC-A2/gp100 and more efficiently than 50 µg Gp100 peptide admixed with CpG [35 ].

It is noteworthy that exosomes secreted from virally infected BM-DC are immunogenic and capable of priming virus-specific CD4+ and CD8+ T cells, protecting the hosts against virus-induced tumors (C. Melief, personal communication). Therefore, mouse Dex can present endogenous antigens harbored by DC. This notion was first described by Aline and colleagues [36 ], demonstrating that Toxoplasma gondii-pulsed DC secrete exosomes mediating antigen-specific T helper cell type 1 immune responses, protective against infection.

In parallel, the mechanisms by which mouse Dex loaded onto DC mediate efficient priming were assessed. Using mice bearing genetic defects for MFG-E8 or B7 molecules, Segura et al. [26 , 27 ] demonstrated that exosomal MFG-E8 is dispensable for the Dex-mediated CD4+ T cell proliferation in vitro and in vivo. Similarly, exosomal B7 molecules did not play a critical role in contrast to the B7 molecules expressed on the recipient DC. However, and as suggested by Sprent [37 ], exosomal ICAM-1/CD54 molecules appeared indispensable for the efficient triggering of naive T cells in vitro and in vivo [26 , 27 ].

Optimization of T cell priming with imDex
Two strategies have been evaluated: Their efficiency against tumors and their pros and cons for clinical implementations are discussed below.

The first one consisted of promoting DC maturation before exosome purification (mDex); the second one, in neutralizing regulatory T (Treg) cells prior to imDex inoculation.

imBM-DC are those cells that are CD40- and CD80-negative (and express low levels of membrane I-Ab molecules) but have the potential to become CD40+ and CD80+ after LPS stimulation. Segura et al. [26 , 27 ] showed that mDex (released from LPS-activated BM-DC or D1 cells) are produced less abundantly than imDex by DC (average of two- to threefold less amounts of proteins) but are qualitatively enriched in molecules involved in mDex-mediated CD4+ T cell priming such as ICAM-1. mDex could induce potent CD4+T cell proliferation in vitro, 50- to 100-fold more efficient than the one observed with imDex. Moreover, these mDex could induce CD4+T cell priming in vivo with ten- to 20-fold less proteins than imDex [26 , 27 ]. Consequently, mDex could contribute to accelerated, allogeneic skin graft rejection compared with imDex (which did not promote tolerance [26 , 27 ]). In addition, mDex could induce potent stimulation of naive CD8+T cells in vitro (proliferation, cytokine synthesis, and differentiation into CTL) [37 ]. mDex-mediated CD8+T cell activation was dependent on the presence of ICAM-1 and B7 molecules on mDex membranes. All these data suggest that inducing DC maturation may improve the efficiency of Dex for induction of adaptive immune responses.

The role of CD4+CD25+ Treg cells in restricting T cell-based immune responses has gained renewed recognition [38 ]. Treg prevent primary and secondary antitumor T cell responses. Cyclophosphamide (CTX), at immunopotentiating dosages, has been shown to reduce numbers and suppressive functions of Treg [39 40 41 ]. Therefore, we investigated the combined effects of CTX and imDex on poorly immunogenic melanoma. The imDex-mediated antitumor effects were improved dramatically in CTX-pretreated animals bearing already established tumors. Therapy with CTX prior to tumor establishment was not efficient to boost imDex beneficial effects. We showed that vaccination with imDex but not free peptides could promote tumor or peptide-specific secondary responses, only in the absence of Treg. Adoptive transfer of Treg indeed curtailed CTX/imDex synergistic effects, and depletion of natural killer (NK) cells did not prevent the antitumor activity of the association [41 ]. It is important to note that CTX did not allow CTL priming with imDex in the absence of adjuvants but could dramatically boost tumor-induced CD8+ T cell responses. Altogether, imDex represent valuable cell-free peptide vaccines when combined with TLR3/9 ligands or drugs inhibiting Treg cells.

Can Dex mediate T cell tolerance?
Indeed, several reports claim that in some culture conditions, DC release tolerogenic vesicles reducing inflammatory and autoimmune diseases. BM-DC, propagated in recombinant interleukin (rIL)-10 or infected with recombinant adenoviral vectors encoding viral IL-10 or Fas ligand (FasL), could secrete exosomes suppressing delayed-type hypersensitivity (DTH) responses and collagen-induced arthritis following periarticular or systemic inoculation in a MHC Class II and antigen-dependent manner [42 , 43 ]. Moreover, in a rat model of allogeneic heart transplantation, intravenous inoculation of allogenic imDex could delay acute allograft rejection and induce a significant prolongation of allograft survival [44 ].

Likewise, other exosome sources such as intestinal epithelial cells or T and melanoma tumor cells could secrete vesicles capable of inducing antigen-specific tolerance and FasL-mediated T cell apoptosis, respectively [45 46 47 ]. Secretion of exosomal FasL may represent one mechanism by which the placenta is a local immune privilege. Indeed, Frangsmyr et al. [48 ] showed that human syncytiotrophoblast lacks surface expression of FasL but is an early source of the cytoplasmic microvesicular form of FasL.

Induction of innate immunity with Dex
Intriguing and controversial observations tend to suggest that tumor-derived exosomes might interact with NK cells. Using colon and pancreatic tumor cell lines devoid of surface expression of hsp70, Gastpar et al. [49 ] demonstrated that exosomes secreted from those cell lines could promote NK cell chemoattraction and activation (killing of target cells) in a hsp70-dependent manner. Moreover, they went on demonstrating that exosomal hsp72, induced by stimulation of tumor cells with IFN-{gamma}, promotes the up-regulation of CD83 and the IL-12 production on DC [50 ]. However, other investigators have been able to show that tumor exosomes can harbor NKG2D ligands (NKG2DLs) and promote the down-regulation of the expression of the activating NKG2D receptors on CD3– and CD8+ T cells in vitro [51 ].

During the Phase I trial using imDex in Stage IV melanoma patients (see below), the discrepancy between some clinical regressions and the lack of any detectable T cell responses in blood prompted the search for alternate effector mechanisms [52 ]. We studied NK activity in patients enrolled in this clinical trial and could show that Dex were able to restore NK cell activity in seven out of 14 patients analyzed (Chaput et al., unpublished results). To illustrate this new bioactivity of Dex, we report here the case of some patients (Pt#1, Pt#2, Pt#3, and Pt#12) for whom four inoculations of imDex were followed by a significant increase in NK cell activity (Figs. 1 and 2 ). Indeed, NK cell-killing activity against the erythroleukemia K562 and IFN-{gamma} secretion after ex vivo stimulation were enhanced after the treatment in seven out of 15 patients (data from Patients #3 and #12 are shown in Fig. 1 ). In three patients for whom the autologous tumor cell line could be established in vitro, we could show that NK cells became capable of recognizing the autologous tumor cells after therapy with imDex (Fig. 2A) . For patient 12, recruitment and/or trafficking of NK cells inside tumor areas could be documented (Fig. 2B 2C 2D) . Therefore, NK cell activation represents a new pharmacodynamic parameter for Dex bioactivity in patients. The molecular mechanisms of Dex-mediated NK cell triggering are currently under investigation.


Figure 1
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  		Figure 1. Long-term NK cell activation with Dex vaccines in two patients. NK cells at Week 1 (W1; before Dex therapy), W7 (7 weeks after the first Dex administration), and W30 (Dex administration every-other 3 weeks for 6–10 months for patients who benefit from a continuation of Dex therapy) were cocultured alone, in IL-2 (IL-2 was not tested at W30), or with DC in the presence or absence of LPS prior to chromium release assay against K562 (Pt#12, A) or DAUDI (Pt#3, C) at 10:1 (shown) and 2:1 (not shown) effector:target (E:T) ratios. IFN-{gamma} levels were measured in the supernatants of cocultures (B, Pt#12; D, Pt#3). Each experiment was performed three times with similar results. Means ± SEM of specific lysis and IFN-{gamma} of a representative experiment are depicted, and statistical analyses using Fisher’s exact method were performed (*, P<0.05).

 

Figure 2
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  		Figure 2. Dex induced NK cell recognition of autologous tumors. (A) Autologous melanoma cell recognition by NK cells at W1 and W7 for Patients #1, #2, and #12 in a standard chromium release assay at a 20:1 E:T ratio (Patient #2 and Patient #12) and 50:1 E:T ratio (Patient #1) after NK cell purification and overnight stimulation in human rIL-2 (rhuIL-2) 1000 IU/ml. (B–D) Immunohistochemical analyses of the NK cell infiltrates in tumor specimen before and after Dex vaccination for Patient #12. Double-staining with anti-CD3 (red) and anti-CD57 (brown) monoclonal antibodies (mAb) after 10 exosome inoculations at a high-power magnification. NK cells are CD3–CD57+, and double-staining represents activated T cells. (B) Staining with anti-PEN5 (mostly staining CD56dim NK cells) mAb before (C) versus after (D) exosome therapy. This staining allowed the enumeration of tumor-infiltrating NK cells before (210 NK cells/mm2) and after (440 NK cells/mm2) Dex vaccines.

 

   RESULTS OF THE TWO FIRST PHASE I CLINICAL TRIALS
TOP
 ABSTRACT
 INTRODUCTION
 FORMATION AND COMPOSITION OF...
 BIOACTIVITY OF Dex: PRECLINICAL...
 RESULTS OF THE TWO...
CONCLUSIONS AND FUTURE PROSPECTS
 REFERENCES
 
The research and development leading to a clinical implementation were led by Anosys SA Inc. (France), which worked out a closed GMP for exosome purification and defined the quality control criteria for therapeutic imDex preparations [19 , 32 ]. Quality assurance criteria for exosome release and administration in patients were qualitative (expression of CD81 tetraspanins) and quantitative (at least 1x1014 MHC Class II molecules in enzyme-linked immunosorbent assay) and included a functional bioassay using superantigens.

Briefly, therapeutic imDex were purified from autologous DC, propagated from adherent monocytes, incubated in granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 for 5 days. Two Phase I trials were conducted concomitantly: one at the Institut Gustave Roussy and Institut Curie in Paris in melanoma, and a second one, in nonsmall cell lung cancer (NSCLC) at Duke University (Duham, NC). These Phase I trials have investigated Dex safety and surrogate markers of clinical efficacy. Inclusion criteria were leukocyte phenotyping indicating HLA-A1 or B35 and HLA-DP04 and tumor-expressing melanoma antigen gene (MAGE) in Stage IIIB/IV melanoma. In the United States, the Phase I study enrolled HLA A2+ patients with pretreated Stage IIIb (n=4) and IV (n=9) NSCLC with tumor expression of MAGE-A3 or -A4. Both trials assessed autologous imDex purified from MD-DC (GM-CSF+IL-4) loaded with MAGE3DP.04 peptides and after purification, pulsed with MAGE3.A1 peptides (melanoma trial) and MAGE.A3, -A4 peptides (NSCLC trial) and inoculated in 4 weekly subcutaneous/intradermal (s.c./i.d.) injections [52 , 54 ]. Two dose levels of exosomal MHC Class II molecules and MHC Class I peptides pulsed onto imDex were tested. In case of an objective response, the four vaccine injections were followed by boosts every-other 3 weeks (three cases). Secondary endpoints were the immunomonitoring of peptide-specific CD4+ and CD8+ T cell responses restricted by exosomal MHC Classes II and I molecules. Fifteen melanoma patients were included and 13 NSCLC bearing patients.

Trial in metastatic melanoma [52 ]
Leukaphereses of 1.5 blood mass performed in the 15 metastatic melanoma patients enrolled in the study allowed the recovery of 1.93 x 109 CD14+ cells (range: 1.06–4.64). These monocytes differentiated into imMD-DC in rhuGM-CSF and rhuIL-4 (3.28x108, range: 0.2–12 at Day 7). Human DC were considered "immature" when lacking the expression of CD83.

In three patients out of 15, the first leukapheresis did not allow CD14+ cells to adhere, and consequently, a second leukapheresis was required to harvest exosomes. The GMP process allowed to purify ~4.50 x 1014 (range: 0.13–15) exosomal MHC Class II molecules from supernatants of Days 6–7 MD-DC requested for up to 41 (range: 9–115) s.c./i.d. vaccinations/patient at the lowest dose (i.e , 0.13x1014 exosomal MHC Class II molecules/vaccine). As for the patients who benefited from continuation treatment requiring a second (Patients #3, #12, and #14) and eventually, a third leukapheresis (Patient #12), the exosome recovery allowed retreatment in all cases. It is noteworthy that a large variability in the quantity of exosomes (measured as the number of MHC Class II molecules) produced between individual lots was observed (>50-fold) in 144 exosome preparations from normal donors (111) and cancer patients (33). The number of cells collected per individual and the productivity of these cells to differentiate into DC are the principal sources of variability in the production of Class II [54 ].

There was no Grade II toxicity, and no maximal tolerated dose was identified. Five out of 15 patients experienced some clinical benefit (one partial, one minor, two stable, and one mixed responses) in skin and lymph node sites, some of them having experienced vaccination failure with DNA or MAGE protein/peptides. There was no exosome or peptide dose response, and the maximal tolerated dose was not reached. No T cell responses directed against the vaccinating peptides or the tumor cells could be detected in blood after four imDex vaccines. In contrast, vaccination with imDex could significantly promote NK cell effector function in seven of 15 patients, eventually leading to autologous tumor recognition (as developed above).

Trial in NSCLC [53 ]
Nine out of 13 enrolled patients completed the imDex therapy. The mean imDex production was 3.14 x 1014 (range: 4.1x1012–9.1x1014) exosomal MHC Class II molecules. The mean quantity of imDex secreted from MD-DC derived from 111 normal volunteers (NV) was not significantly different, i.e., 3.9 x 1014 total MHC Class II molecules. Only Grades 1–2 adverse events were reported. Survival of patients after the first Dex dose was 52–665+ days. DTH reactivities against MAGE peptides were detected in three of nine patients. MAGE-specific T cell responses were monitored in one of three patients, and NK cell lytic activity was restored for two of four patients.


   CONCLUSIONS AND FUTURE PROSPECTS
TOP
 ABSTRACT
 INTRODUCTION
 FORMATION AND COMPOSITION OF...
 BIOACTIVITY OF Dex: PRECLINICAL...
 RESULTS OF THE TWO...
 CONCLUSIONS AND FUTURE PROSPECTS
REFERENCES
 
Although cell therapy remains a controversial anticancer strategy, accumulating evidence points to a role of the immune system, de novo or boosted by therapies, in disease-free survival in cancer-bearing patients [55 56 57 ]. Moreover, some cytotoxic agents can mediate part of their antitumor activity through the immune system [58 ] and/or inhibit Treg cells. Therefore, immunotherapy, in conjunction with conventional cytotoxic agents, will become part of the oncologist armamentarium to counterbalance tumor-induced tolerance.

The feasability of Dex production in advanced cancer patients together with the lack of toxicity, the novel bioactivity on NK cells, and the interesting clinical observations of long-term stability support further investigations of Dex immunotherapy.

Several points deserve to be underscored
First, like alternate immunotherapeutic approaches, surrogate endpoints for therapy with Dex should be progression-free survival and not objective responses, and to that extent, the NSCLC model represents a more suitable cancer for a randomized Phase III study than melanoma.

Second, the Dex vaccine will be optimal if Dex express ICAM-1 molecules, if host DC bear CD86 molecules (activated status), and Treg are suppressed at the start. The CTX dosing for elective depletion and inhibition of Treg has been set up by F. Ghiringhelli et al. (unpublished results).

MD-DC should be propagated in GM-CSF + IL-4 and activated during exosome collection (mDex) with a TLR3 agonist in vitro for 24 h. This TLR3 agonist should also be administered systemically in patients, concomittantly to i.d. injection of mDex. TLR3 ligands of GMP grade are currently being used in the clinic [59 ]. Four weekly vaccines will be followed by boost with mDex every 3 weeks in the absence of adjuvant.

Third, our unpublished data point to the capacity of imDex to elicit potent, non-MHC-restricted, NKG2D-dependent T and NK cell responses, which could modulate the tumor microenvironment and/or the lymph node areas. Therefore, our favorite surrogate markers will be NKG2D expression and functions and NKG2D-dependent CD8 and NK cell activation.

Finally, along with the classical T cell follow-up (including Treg cells), we need to investigate MHC Classes I and II, killer inhibitory receptors, NKG2D, NKp30 genetic polymorphisms, as well as the tumor profiling (including NKG2DLs) [60 ].

Consequently, we are planning to conduct a Phase III clinical trial in advanced HLA-A2 + NSCLC Stage IIIB/IV patients. Sixty patients will be randomized after stabilization or regression with three cycles of conventionnal chemotherapy between CTX and CTX + TLR3 agonist + mDex.


   ACKNOWLEDGEMENTS
 
This work was supported from the Cancéropôle Idf, by INCa, and by the European community QLRT-2001-00093.

Received February 13, 2006; revised May 16, 2006; accepted May 17, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 FORMATION AND COMPOSITION OF...
 BIOACTIVITY OF Dex: PRECLINICAL...
 RESULTS OF THE TWO...
 CONCLUSIONS AND FUTURE PROSPECTS
 REFERENCES
 

  1. Fevrier, B., Raposo, G. (2004) Exosomes: endosomal-derived vesicles shipping extracellular messages Curr. Opin. Cell Biol. 16,415-421[CrossRef][Medline]
  2. Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J., Raposo, G. (2002) The biogenesis and functions of exosomes Traffic 3,321-330[CrossRef][Medline]
  3. Thery, C., Zitvogel, L., Amigorena, S. (2002) Exosomes: composition, biogenesis and function Nat. Rev. Immunol. 2,569-579[Medline]
  4. Johnstone, R. M., Adam, M., Hammond, J. R., Orr, L., Turbide, C. (1987) Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes) J. Biol. Chem. 262,9412-9420[Abstract/Free Full Text]
  5. Johnstone, R. M., Bianchini, A., Teng, K. (1989) Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions Blood 74,1844-1851[Abstract/Free Full Text]
  6. Johnstone, R. M., Ahn, J. (1990) A common mechanism may be involved in the selective loss of plasma membrane functions during reticulocyte maturation Biomed. Biochim. Acta 49,S70-S75[Medline]
  7. Johnstone, R. M., Mathew, A., Mason, A. B., Teng, K. (1991) Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins J. Cell. Physiol. 147,27-36[CrossRef][Medline]
  8. Blanchard, N., Lankar, D., Faure, F., Regnault, A., Dumont, C., Raposo, G., Hivroz, C. (2002) TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/{zeta} complex J. Immunol. 168,3235-3241[Abstract/Free Full Text]
  9. Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., Flament, C., Tenza, D., Ricciardi-Castagnoli, P., Raposo, G., Amigorena, S. (1998) Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes Nat. Med. 4,594-600[CrossRef][Medline]
  10. Raposo, G., Nijman, H. W., Stoorvogel, W., Liejendekker, R., Harding, C. V., Melief, C. J., Geuze, H. J. (1996) B lymphocytes secrete antigen-presenting vesicles J. Exp. Med. 183,1161-1172[Abstract/Free Full Text]
  11. van Niel, G., Heyman, M. (2002) The epithelial cell cytoskeleton and intracellular trafficking. II. Intestinal epithelial cell exosomes: perspectives on their structure and function Am. J. Physiol. Gastrointest. Liver Physiol. 283,G251-G255[Abstract/Free Full Text]
  12. Van Niel, G., Mallegol, J., Bevilacqua, C., Candalh, C., Brugiere, S., Tomaskovic-Crook, E., Heath, J. K., Cerf-Bensussan, N., Heyman, M. (2003) Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice Gut 52,1690-1697[Abstract/Free Full Text]
  13. Taylor, D. D., Gercel-Taylor, C. (2005) Tumor-derived exosomes and their role in cancer-associated T-cell signaling defects Br. J. Cancer 92,305-311[Medline]
  14. Wolfers, J., Lozier, A., Raposo, G., Regnault, A., Thery, C., Masurier, C., Flament, C., Pouzieux, S., Faure, F., Tursz, T., Angevin, E., Amigorena, S., Zitvogel, L. (2001) Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming Nat. Med. 7,297-303[CrossRef][Medline]
  15. Riteau, B., Faure, F., Menier, C., Viel, S., Carosella, E. D., Amigorena, S., Rouas-Freiss, N. (2003) Exosomes bearing HLA-G are released by melanoma cells Hum. Immunol. 64,1064-1072[CrossRef][Medline]
  16. Andre, F., Schartz, N. E., Chaput, N., Flament, C., Raposo, G., Amigorena, S., Angevin, E., Zitvogel, L. (2002) Tumor-derived exosomes: a new source of tumor rejection antigens Vaccine 20(Suppl. 4),A28-A31[Medline]
  17. Andre, F., Schartz, N. E., Movassagh, M., Flament, C., Pautier, P., Morice, P., Pomel, C., Lhomme, C., Escudier, B., Le Chevalier, T., Tursz, T., Amigorena, S., Raposo, G., Angevin, E., Zitvogel, L. (2002) Malignant effusions and immunogenic tumor-derived exosomes Lancet 360,295-305[CrossRef][Medline]
  18. Delcayre, A., Le Pecq, J. B. (2006) Exosomes as novel therapeutic nanodevices Curr. Opin. Mol. Ther. 8,31-38[Medline]
  19. Lamparski, H. G., Metha-Damani, A., Yao, J. Y., Patel, S., Hsu, D. H., Ruegg, C., Le Pecq, J. B. (2002) Production and characterization of clinical grade exosomes derived from dendritic cells J. Immunol. Methods 270,211-226[CrossRef][Medline]
  20. Thery, C., Regnault, A., Garin, J., Wolfers, J., Zitvogel, L., Ricciardi-Castagnoli, P., Raposo, G., Amigorena, S. (1999) Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73 J. Cell Biol. 147,599-610[Abstract/Free Full Text]
  21. Thery, C., Boussac, M., Veron, P., Ricciardi-Castagnoli, P., Raposo, G., Garin, J., Amigorena, S. (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles J. Immunol. 166,7309-7318[Abstract/Free Full Text]
  22. Clayton, A., Court, J., Navabi, H., Adams, M., Mason, M. D., Hobot, J. A., Newman, G. R., Jasani, B. (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry J. Immunol. Methods 247,163-174[CrossRef][Medline]
  23. Hemler, M. E. (2003) Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain Annu. Rev. Cell Dev. Biol. 19,397-422[CrossRef][Medline]
  24. Clayton, A., Harris, C. L., Court, J., Mason, M. D., Morgan, B. P. (2003) Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59 Eur. J. Immunol. 33,522-531[CrossRef][Medline]
  25. Katzmann, D. J., Odorizzi, G., Emr, S. D. (2002) Receptor downregulation and multivesicular-body sorting Nat. Rev. Mol. Cell Biol. 3,893-905[CrossRef][Medline]
  26. Segura, E., Amigorena, S., Thery, C. (2005) Mature dendritic cells secrete exosomes with strong ability to induce antigen-specific effector immune responses Blood Cells Mol. Dis. 35,89-93[CrossRef][Medline]
  27. Segura, E., Nicco, C., Lombard, B., Veron, P., Raposo, G., Batteux, F., Amigorena, S., Thery, C. (2005) ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming Blood 106,216-223[Abstract/Free Full Text]
  28. Silvestre, J. S., Thery, C., Hamard, G., Boddaert, J., Aguilar, B., Delcayre, A., Houbron, C., Tamarat, R., Blanc-Brude, O., Heeneman, S., Clergue, M., Duriez, M., Merval, R., Levy, B., Tedgui, A., Amigorena, S., Mallat, Z. (2005) Lactadherin promotes VEGF-dependent neovascularization Nat. Med. 11,499-506[CrossRef][Medline]
  29. Laulagnier, K., Motta, C., Hamdi, S., Roy, S., Fauvelle, F., Pageaux, J. F., Kobayashi, T., Salles, J. P., Perret, B., Bonnerot, C., Record, M. (2004) Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization Biochem. J. 380,161-171[CrossRef][Medline]
  30. Laulagnier, K., Grand, D., Dujardin, A., Hamdi, S., Vincent-Schneider, H., Lankar, D., Salles, J. P., Bonnerot, C., Perret, B., Record, M. (2004) PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes FEBS Lett. 572,11-14[CrossRef][Medline]
  31. Laulagnier, K., Vincent-Schneider, H., Hamdi, S., Subra, C., Lankar, D., Record, M. (2005) Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids Blood Cells Mol. Dis. 35,116-121[CrossRef][Medline]
  32. Hsu, D. H., Paz, P., Villaflor, G., Rivas, A., Mehta-Damani, A., Angevin, E., Zitvogel, L., Le Pecq, J. B. (2003) Exosomes as a tumor vaccine: enhancing potency through direct loading of antigenic peptides J. Immunother. 26,440-450[Medline]
  33. Thery, C., Duban, L., Segura, E., Veron, P., Lantz, O., Amigorena, S. (2002) Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes Nat. Immunol. 3,1156-1162[CrossRef][Medline]
  34. Andre, F., Chaput, N., Schartz, N. E., Flament, C., Aubert, N., Bernard, J., Lemonnier, F., Raposo, G., Escudier, B., Hsu, D. H., Tursz, T., Amigorena, S., Angevin, E., Zitvogel, L. (2004) Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells J. Immunol. 172,2126-2136[Abstract/Free Full Text]
  35. Chaput, N., Schartz, N. E., Andre, F., Taieb, J., Novault, S., Bonnaventure, P., Aubert, N., Bernard, J., Lemonnier, F., Merad, M., Adema, G., Adams, M., Ferrantini, M., Carpentier, A. F., Escudier, B., Tursz, T., Angevin, E., Zitvogel, L. (2004) Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection J. Immunol. 172,2137-2146[Abstract/Free Full Text]
  36. Aline, F., Bout, D., Amigorena, S., Roingeard, P., Dimier-Poisson, I. (2004) Toxoplasma gondii antigen-pulsed-dendritic cell-derived exosomes induce a protective immune response against T. gondii infection Infect. Immun. 72,4127-4137[Abstract/Free Full Text]
  37. Sprent, J. (2005) Direct stimulation of naive T cells by antigen-presenting cell vesicles Blood Cells Mol. Dis. 35,17-20[CrossRef][Medline]
  38. Curiel, T. J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., Evdemon-Hogan, M., Conejo-Garcia, J. R., Zhang, L., Burow, M., et al (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival Nat. Med. 10,942-949[CrossRef][Medline]
  39. Ghiringhelli, F., Larmonier, N., Schmitt, E., Parcellier, A., Cathelin, D., Garrido, C., Chauffert, B., Solary, E., Bonnotte, B., Martin, F. (2004) CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative Eur. J. Immunol. 34,336-344[CrossRef][Medline]
  40. Lutsiak, M. E., Semnani, R. T., De Pascalis, R., Kashmiri, S. V., Schlom, J., Sabzevari, H. (2005) Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide Blood 105,2862-2868[Abstract/Free Full Text]
  41. Taieb, J., Chaput, N., Schartz, N., Roux, S., Novault, S., Menard, C., Ghiringhelli, F., Terme, M., Carpentier, A. F., Darrasse-Jese, G., Lemonnier, F., Zitvogel, L. (2006) Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines J. Immunol. 176,2722-2729[Abstract/Free Full Text]
  42. Kim, S. H., Lechman, E. R., Bianco, N., Menon, R., Keravala, A., Nash, J., Mi, Z., Watkins, S. C., Gambotto, A., Robbins, P. D. (2005) Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis J. Immunol. 174,6440-6448[Abstract/Free Full Text]
  43. Kim, S. H., Bianco, N., Menon, R., Lechman, E. R., Shufesky, W. J., Morelli, A. E., Robbins, P. D. (2006) Exosomes derived from genetically modified DC expressing FasL are anti-inflammatory and immunosuppressive Mol. Ther. 13,289-300[CrossRef][Medline]
  44. Peche, H., Heslan, M., Usal, C., Amigorena, S., Cuturi, M. C. (2003) Presentation of donor major histocompatibility complex antigens by bone marrow dendritic cell-derived exosomes modulates allograft rejection Transplantation 76,1503-1510[CrossRef][Medline]
  45. Andreola, G., Rivoltini, L., Castelli, C., Huber, V., Perego, P., Deho, P., Squarcina, P., Accornero, P., Lozupone, F., Lugini, L., Stringaro, A., Molinari, A., Arancia, G., Gentile, M., Parmiani, G., Fais, S. (2002) Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles J. Exp. Med. 195,1303-1316[Abstract/Free Full Text]
  46. Karlsson, M., Lundin, S., Dahlgren, U., Kahu, H., Pettersson, I., Telemo, E. (2001) "Tolerosomes" are produced by intestinal epithelial cells Eur. J. Immunol. 31,2892-2900[CrossRef][Medline]
  47. Martinez-Lorenzo, M. J., Anel, A., Gamen, S., Monlen, n., , I., Lasierra, P., Larrad, L., Pineiro, A., Alava, M. A., Naval, J. (1999) Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles J. Immunol. 163,1274-1281[Abstract/Free Full Text]
  48. Frangsmyr, L., Baranov, V., Nagaeva, O., Stendahl, U., Kjellberg, L., Mincheva-Nilsson, L. (2005) Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level Mol. Hum. Reprod. 11,35-41[Abstract/Free Full Text]
  49. Gastpar, R., Gehrmann, M., Bausero, M. A., Asea, A., Gross, C., Schroeder, J. A., Multhoff, G. (2005) Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells Cancer Res. 65,5238-5247[Abstract/Free Full Text]
  50. Bausero, M. A., Gastpar, R., Multhoff, G., Asea, A. (2005) Alternative mechanism by which IFN-{gamma} enhances tumor recognition: active release of heat shock protein 72 J. Immunol. 175,2900-2912[Abstract/Free Full Text]
  51. Clayton, A., Tabi, Z. (2005) Exosomes and the MICA-NKG2D system in cancer Blood Cells Mol. Dis. 34,206-213[CrossRef][Medline]
  52. Escudier, B., Dorval, T., Chaput, N., Andre, F., Caby, M. P., Novault, S., Flament, C., Leboulaire, C., Borg, C., Amigorena, S., et al (2005) Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial J. Transl. Med. 3,10[CrossRef][Medline]
  53. Morse, M. A., Garst, J., Osada, T., Khan, S., Hobeika, A., Clay, T. M., Valente, N., Shreeniwas, R., Sutton, M. A., Delcayre, A., Hsu, D. H., Le Pecq, J. B., Lyerly, H. K. (2005) A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer J. Transl. Med. 3,9[CrossRef][Medline]
  54. Patel, S., Mehta-Damani, A., Shu, H., Le Pecq, J. B. (2005) An analysis of variability in the manufacturing of dexosomes: implications for development of an autologous therapy Biotechnol. Bioeng. 92,238-249[CrossRef][Medline]
  55. Borg, C., Terme, M., Taieb, J., Menard, C., Flament, C., Robert, C., Maruyama, K., Wakasugi, H., Angevin, E., Thielemans, K., et al (2004) Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell-dependent antitumor effects J. Clin. Invest. 114,379-388[CrossRef][Medline]
  56. Pages, F., Berger, A., Camus, M., Sanchez-Cabo, F., Costes, A., Molidor, R., Mlecnik, B., Kirilovsky, A., Nilsson, M., Damotte, D., Meatchi, T., Bruneval, P., Cugnenc, P. H., Trajanoski, Z., Fridman, W. H., Galon, J. (2005) Effector memory T cells, early metastasis, and survival in colorectal cancer N. Engl. J. Med. 353,2654-2666[Abstract/Free Full Text]
  57. Zhang, L., Conejo-Garcia, J. R., Katsaros, D., Gimotty, P. A., Massobrio, M., Regnani, G., Makrigiannakis, A., Gray, H., Schlienger, K., Liebman, M. N., Rubin, S. C., Coukos, G. (2003) Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer N. Engl. J. Med. 348,203-213[Abstract/Free Full Text]
  58. Casares, N., Pequignot, M. O., Tesniere, A., Ghiringhelli, F., Roux, S., Chaput, N., Schmitt, E., Hamai, A., Hervas-Stubbs, S., Obeid, M., Coutant, F., Metivier, D., Pichard, E., Aucouturier, P., Pierron, G., Garrido, C., Zitvogel, L., Kroemer, G. (2005) Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death J. Exp. Med. 202,1691-1701[Abstract/Free Full Text]
  59. Suhadolnik, R. J., Reichenbach, N. L., Hitzges, P., Adelson, M. E., Peterson, D. L., Cheney, P., Salvato, P., Thompson, C., Loveless, M., Muller, W. E., et al (1994) Changes in the 2-5A synthetase/RNase L antiviral pathway in a controlled clinical trial with poly(I)-poly(C12U) in chronic fatigue syndrome In Vivo 8,599-604[Medline]
  60. Hayashi, T., Imai, K., Morishita, Y., Hayashi, I., Kusunoki, Y., Nakachi, K. (2006) Identification of the NKG2D haplotypes associated with natural cytotoxic activity of peripheral blood lymphocytes and cancer immunosurveillance Cancer Res. 66,563-570[Abstract/Free Full Text]



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S. Bhatnagar, K. Shinagawa, F. J. Castellino, and J. S. Schorey
Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo
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