Julien Taieb

A novel dendritic cell subset involved in tumor immunosurveillance

The interferon (IFN)-γ–induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-γ is not the conventional NK cell but a subset of B220+Ly6C− dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220+NK1.1+ dendritic cells secrete high levels of IFN-γ and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2−/−Il2rg−/− mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response.

Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell–dependent antitumor effects

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.

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.

Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines.

Dendritic cell-derived exosomes (DEX) are nanomeric vesicles harboring MHC/peptide complexes capable of promoting primary T cell responses and tumor rejection in the presence of adjuvants. In this study, we show that, in the absence of adjuvants, DEX mediate potent Ag-dependent antitumor effects against preestablished tumors in mice pretreated with immunopotentiating dosing of cyclophosphamide. Cyclophosphamide could 1) abolish the suppressive function of CD4+CD25+Foxp3+ regulatory T cells, 2) markedly enhance the magnitude of secondary but not primary CTL responses induced by DEX vaccines, 3) synergize with DEX in therapy but not prophylaxis tumor models. Therefore, therapeutic vaccines such as DEX aimed at boosting tumor-primed effector T cells could benefit procedures that minimize the effects of CD4+CD25+ regulatory T cells.

Dendritic Cell-Derived Exosomes Promote Natural Killer Cell Activation and Proliferation: A Role for NKG2D Ligands and IL-15Rα

Dendritic cell (DC) derived-exosomes (Dex) are nanomeric vesicles harboring functional MHC/peptide complexes promoting T cell-dependent tumor rejection. In the first Phase I trial using peptide-pulsed Dex, the observation of clinical regressions in the absence of T cell responses prompted the search for alternate effector mechanisms. Mouse studies unraveled the bioactivity of Dex on NK cells. Indeed, Dex promoted an IL-15Rα- and NKG2D-dependent NK cell proliferation and activation respectively, resulting in anti-metastatic effects mediated by NK1.1+ cells. In humans, Dex express functional IL-15Rα which allow proliferation and IFNγ secretion by NK cells. In contrast to immature DC, human Dex harbor NKG2D ligands on their surface leading to a direct engagement of NKG2D and NK cell activation ex vivo. In our phase I clinical trial, we highlight the capacity of Dex based-vaccines to restore the number and NKG2D-dependent function of NK cells in 7/14 patients. Altogether, these data provide a mechanistic explanation on how Dex may stimulate non MHC restricted-anti-tumor effectors and induce tumor regression in vivo.

Truth survival in clinical research: An evidence-based requiem?

Science progresses through a series of paradigms that are held to be true until they are replaced by a better approximation of reality (1). Since the development of the steam engine in the late 18th century, economists have recognized 50-year cycles during which critical technological innovation is introduced (2). In 1997, Hall and Platell (3) estimated the half-life of dogma relating to the practice of surgery. From their analysis of 260 abstracts published from 1935 to 1994, they estimated that the half-life of truth for clinical conclusions in the surgical literature was 45 years. We hypothesized that some factors should be related to this truth survival. The first hypothesis was that conclusions derived from better methodology should have a longer half-life. If correct, this observation could be a validation of good methodology, often called evidence-based medicine (4). Therefore, we compared the survival of conclusions from meta-analyses with those from isolated, randomized trials or nonrandomized studies. For the conclusions from randomized trials (isolated trials or meta-analyses), we also compared the survival rate on the basis of high versus low methodologic scores. The second hypothesis was that survival of truth should be higher for negative conclusions than for positive conclusions. A negative conclusion has a better chance of survival because the only way it would not continue to be negative is if it were found to be false. A positive conclusion risks being found to be false or becoming obsolete. We also thought that publication of a negative conclusion in a reputable journal often indicated that a previous positive conclusion had been found to be false. We concluded, therefore, that this second-line analysis should be of higher quality. To reduce heterogeneity in sampling and evaluation, we chose a single medical disciplinecirrhosis and hepatitisand focused on two selective journals. We tried to categorize the conclusions into three groups: those that were true (referred to as true in this article), those that were not false but became obsolete (referred to as obsolete), and those that are now considered false (referred to as false). An example of an obsolete conclusion is the efficacy of immunoglobulins for preventing hepatitis A virus infection, since an effective vaccine is now available. An example of a false conclusion is the efficacy of corticosteroids for treating acute viral hepatitis. Methods We identified original articles about cirrhosis or hepatitis in adults from 1945 to 1999. The articles were divided into eleven 5-year periods. Nonoriginal studies and studies involving children were excluded. Selection of Nonrandomized Studies In each 5-year period, we selected 20 nonrandomized articles published in two journals10 from Lancet and 10 from Gastroenterology. We chose these journals because they have published clinical studies about hepatitis and cirrhosis since at least 1945, they are peer-reviewed and highly selective, and they have impact factors greater than 10. Articles from 1945 to 1985 were selected by a hand search. Because a true randomization was very difficult to organize, we selected by order of publication within each 5-year period. We selected the first article about cirrhosis or hepatitis that appeared within each 5-year period, then the last published article in the period, then the second article after the first, then the second-to-the-last article, and so on up to 20 articles. From 1985 to 1999, we used a PubMed electronic search and specified the following limits: cirrhosis or hepatitis, human, and Lancet or Gastroenterology. Abstracts were downloaded by using a similar selection method, stratified into 5-year periods. We selected the first abstract listed on the first electronic page, then the first on the last electronic page, then the last on the second electronic page, then the last on the next-to-the- last electronic page, and so on up to 20 articles. Selection of Randomized Trials In each 5-year period, we tried to select 20 randomized trials about cirrhosis or hepatitis, 10 from Lancet and 10 from Gastroenterology. This was possible from 1970 to 1999. From 1945 to 1969, we selected all randomized trials that could be identified in any journal (range, 4 trials [1945 to 1950] to 20 trials [1965 to 1969]). From 1945 to 1982, we used the hand searching method previously described (5). We completed the random selection by hand searching articles from 1982 to 1985 and using PubMed (as described for nonrandomized studies) to search for articles from 1985 to 1999. Selection of Meta-Analyses From 1945 to 1992, we used a hand-searching method, as described in a systematic review of meta-analyses (6). Thereafter, we used PubMed and specified the following limits: meta-analysis and cirrhosis or hepatitis. Because of the limited number of meta-analyses, we included all journals. All of the meta-analyses consisted solely of randomized trials. Database Development and Observer Review We obtained abstracts from all of the articles and selected the sentence from each abstract that seemed to best summarize the findings. These sentences were then copied to a database. Editing of these sentences was restricted to the rephrasing of outdated terminology and the elimination of redundant words. Six hepatologists, called observers, assessed a form that contained the selected conclusion sentences in a random order. Observers were full-time hepatologists with different clinical subspecialties (viral hepatitis, n = 2; HIV, n = 1; fibrosis, n = 1; alcoholic liver disease, n = 1; and transplantation, n = 1); worked in the same hospital; and were between 31 and 65 years of age. They had graduated from six different universities; three had worked in the United States, each in a different university. Observers were blinded to period, journal, authors, method (meta-analysis, randomized trial, or nonrandomized study), and the methodologic quality from which each conclusion was derived. They classified each conclusion into one of three categories: still true in 2000, obsolete but not false in 2000, or false in 2000. Quality Assessment of Methodology and Consideration of Prognostic Factors Independent of this study, the quality of the randomized trials was assessed by means of a scoring method (range, 2 to 14; mean, 12) that included 14 items (7, 8). Also independent of this study, the quality of the meta-analyses was assessed by means of a slightly modified version (6) of the scoring method established by Sacks and coworkers (9) and described in detail elsewhere (7). This scoring method (range, 0 to 54; mean, 27) included 27 items. We analyzed the meta-analyses that combined individual data as a separate category of research. In classic meta-analysis, the results of each trial are combined. In meta-analysis that combines individual data, the results for each patient are combined with the patients prognostic factors, thus permitting better adjustment of the treatment effect on prognostic factors. Articles were rated as high quality when the score was greater or equal to the mean (12 for randomized trials and 27 for meta-analyses) and as low quality when the score was less than the mean. The methodologic quality of nonrandomized studies was classified as low because no specific scoring method was available. In addition to methodologic quality, the following factors were considered: negative or positive conclusion, type of disease (hepatitis, portal hypertension, alcoholic liver disease, primary biliary cirrhosis, or miscellaneous), domain of clinical research (therapeutic, diagnostic, or cognitive study [cognitive studies were defined as explanatory studies that did not assess treatment or diagnostic tests]), journal of publication (Lancet, Gastroenterology, or other), and specialty (medicine or surgery). Statistical Analysis A conclusion was considered to be true, obsolete, or false when three or more observers stated it to be so. When there was a split decision (3 to 3) about whether conclusions were true or not true (9 of 474 articles [1.9%]), the conclusion was considered to be true. When there was a split decision (3 to 3) about whether conclusions were obsolete or not obsolete (26 of 474 articles [5.5%]), the conclusion was considered to be obsolete. When the article was not classified as either true or obsolete, it was considered to be false. Conclusions from older research are at greater risk for being refuted or becoming obsolete than are conclusions from more recent studies. Because the end points were highly time dependent, we used time-dependent analyses. The half-life was calculated according to the KaplanMeier method, using the censored time as the duration between the year of publication and the year 2000. The censored time is the time at risk for being refuted or found obsolete. For example, an article published in 1950 had a censored time of 50 years. First, we analyzed the truth survival: If the conclusion was assessed to be true, it was censored at 50 years. If the conclusion was assessed to be false or obsolete, it was considered a failure (death of truth). Second, we analyzed the nonfalse survival; true or obsolete conclusions were considered censored at 50 years. If the conclusion was false, it was considered a failure (death of nonfalse). The factors were compared by using the two-sided log-rank test and the multivariate proportional hazards regression analysis. Agreement among observers was analyzed by using statistics. Results Characteristics of the 474 identified articles are given in Table 1. All nonrandomized studies were published in Lancet (50%) or Gastroenterology (50%); randomized trials were published in Lancet (29%), Gastroenterology (41%), or other journals (30%); and 92% of meta-analyses were published in other journals. Compared with the total number of articles published every year about hepatitis or cirrhosis, this sample represents less than 0.1% of nonran

Dendritic cell derived-exosomes : biology and clinical implementations

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.

Blood neutrophil functions and cytokine release in severe alcoholic hepatitis : effect of corticosteroids

BACKGROUND/AIMSnSeveral observations point to an important role of interactions between polymorphonuclear neutrophils and cytokines in severe alcoholic hepatitis. The polymorphonuclear neutrophil activation status and the local and systemic pro- and anti-inflammatory cytokine responses were quantified. The effect of corticosteroids, widely used in this setting, was evaluated using these parameters.nnnMETHODSnWe studied blood polymorphonuclear neutrophil functions in terms of L-selectin and beta2-integrin expression, H2O2 production and IL-8 and tumor necrosis factor alpha synthesis capacity. We also measured IL-8, tumor necrosis factor alpha and IL-10 plasma and liver tissue levels. Fifteen patients with alcoholic hepatitis were compared to 15 patients with alcoholic cirrhosis without alcoholic hepatitis, and to 10 healthy volunteers. The impact of a 28-day course of corticosteroids on blood neutrophils activation status and cytokine levels was evaluated in patients with alcoholic hepatitis.nnnRESULTSnBlood polymorphonuclear neutrophils were activated, as shown by increased H2O2 production (48+/-6 vs 29+/-6 MFI in healthy controls), and decreased L-selectin expression (300+/-61 vs 449+/-59 in healthy controls). Upon stimulation, polymorphonuclear neutrophils synthesized large amounts of IL-8 (21.7+/-9.2 ng/ml vs 8.8+/-10 ng/ml in healthy controls) and tumor necrosis factor alpha (524+/-132 pg/ml vs 79+/-144 pg/ml in healthy controls). Tumor necrosis factor alpha and IL-8 plasma and tissue levels were markedly increased as IL-10 was barely detectable in alcoholic hepatitis patients, compared to cirrhotic patients and healthy controls. During steroid therapy, plasma levels of the pro-inflammatory cytokine IL-8 fell as early as day 14, while levels of the anti-inflammatory cytokine IL-10 increased on day 21. Finally, polymorphonuclear neutrophil functions returned to normal after treatment.nnnCONCLUSIONnSevere alcoholic hepatitis appears to be associated with polymorphonuclear neutrophil activation and an imbalance between pro- and anti-inflammatory cytokines; during steroid therapy a normalization of these parameters was observed.

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.

The Critical Role of IL-15 in the Antitumor Effects Mediated by the Combination Therapy Imatinib and IL-2

The synergistic antitumor effects of the combination therapy imatinib mesylate (IM) and IL-2 depended upon NK1.1- expressing cells and were associated with the accumulation of CD11cintB220+NK1.1+ IFN-producing killer dendritic cells (IKDC) into tumor beds. In this study, we show that the antitumor efficacy of the combination therapy was compromised in IL-15 and IFN-type 1R loss-of-function mice. IL-15Rα was required for the proliferation of IKDC during IM plus IL-2 therapy. Trans-presentation of IL-15/IL-15Rα activated IKDC to express CCR2 and to respond to type 1 IFN by producing CCL2. Moreover, the antitumor effects of the combination therapy correlated with a CCL2-dependent recruitment of IKDC, but not B220− NK cells, into tumor beds. Altogether, the IL-15-driven peripheral expansion and the CCL-2-dependent intratumoral chemoattraction of IKDC are two critical parameters dictating the antitumor efficacy of IM plus IL-2 in mice.