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Background Microbiome studies report low gut microbial richness and diversity in ulcerative colitis (UC) patients. We explored whether UC patients who reach long-term clinical, endoscopic, and histological remission show a gut microbial ecosystem that is similar to healthy individuals. Methods We collected 184 stool samples from 111 individuals (UC patients in long remission, short remission, flare, and healthy control subjects). Microbiota was analyzed by amplicon sequencing (16S ribosomal RNA) and quantitative polymerase chain reaction for specific taxa. All UC remission patients were followed-up for 2 years. Findings A drop in species diversity and richness, underrepresentation of butyrate producers, and gain of potentially harmful bacteria were significantly detected in samples from disease-flare and short-remission patients. In contrast, Chao1 and Shannon indexes of diversity did not differ among patients in long remission and healthy control subjects. Long-remission patients also presented fecal bacterial composition closer to that in healthy control subjects. There was a positive correlation between Akkermansia muciniphila abundance and time in remission (rs = 0.53, P Interpretation UC patients who achieve long-term remission show evidence of substantial recovery of the gut microbial ecosystem in terms of diversity and composition. Recovery may just reflect adequate control of inflammatory activity, but higher bacterial diversity or the presence of A. muciniphila in fecal samples predicts flare-free outcomes.

International audience; Vectorized small interfering RNAs (siRNAs) are widely used to induce gene silencing. Among the delivery systems used, lipid-based particles are the most effective. Our objective was the development of novel lipid-polymer hybrid nanoparticles, from lipoplexes (complexes of cationic lipid and siRNAs), and poly (lactic-co-glycolic acid) (PLGA), using a simple modified nanoprecipitation method. Due to their morphology, we called these hybrid nanoparticles Spheroplexes. We elucidated their structure using several physico-chemical techniques and showed that they are composed of a hydrophobic PLGA matrix, surrounded by a lipid envelope adopting a lamellar structure, in which the siRNA is complexed, and they retain surface characteristics identical to the starting nanoparticles, i.e. lipoplexes siRNA. We analyzed the composition of the particle population and determined the final percentage of spheroplexes within this population, 80 to 85% depending on the preparation conditions, using fluorescent markers and the ability of flow cytometry to detect nanometric particles (approximately 200 nm). Finally, we showed that spheroplexes are very stable particles and more efficient than siRNA lipoplexes for the delivery of siRNA to cultured cells. We administered spheroplexes contain siRNAs targeting TNF-α to mice with ulcerative colitis induced by dextran sulfate and our results indicate a disease regression effect with a response probably mediated by their uptake by macrophages / monocytes at the level of lamina propria of the colon. The efficacy of decreased level of TNF-α in vivo seemed to be an association of spheroplexes polymer-lipid composition and the specific siRNA. These results demonstrate that spheroplexes are a promising hybrid nanoparticle for the oral delivery of siRNA to the colon.

During this last decade, adoptive transfer of T lymphocytes genetically modified to express chimeric antigen receptors (CARs) emerged as a valuable therapeutic strategy in hematological cancers. However, this immunotherapy has demonstrated limited efficacy in solid tumors. The main obstacle encountered by CAR-T cells in solid malignancies is the immunosuppressive tumor microenvironment (TME). The TME impedes tumor trafficking and penetration of T lymphocytes and installs an immunosuppressive milieu by producing suppressive soluble factors and by overexpressing negative immune checkpoints. In order to overcome these hurdles, new CAR-T cells engineering strategies were designed, to potentiate tumor recognition and infiltration and anti-cancer activity in the hostile TME. In this review, we provide an overview of the major mechanisms used by tumor cells to evade immune defenses and we critically expose the most optimistic engineering strategies to make CAR-T cell therapy a solid option for solid tumors.

Malaltia inflamatòria intestinal; Càrrega microbiana; Predicció Enfermedad inflamatoria intestinal; Carga microbiana; Predicción Inflammatory bowel disease; Microbial load; Prediction Microbiome sequence data have been used to characterize Crohn's disease (CD) and ulcerative colitis (UC). Based on these data, we have previously identified microbiomarkers at the genus level to predict CD and CD relapse. However, microbial load was underexplored as a potential biomarker in inflammatory bowel disease (IBD). Here, we sought to study the use of fungal and bacterial loads as biomarkers to detect both CD and UC and CD and UC relapse. We analyzed the fecal fungal and bacterial loads of 294 stool samples obtained from 206 participants using real-time PCR amplification of the ITS2 region and the 16S rRNA gene, respectively. We combined the microbial data with demographic and standard laboratory data to diagnose ileal or ileocolonic CD and UC and predict disease relapse using the random forest algorithm. Fungal and bacterial loads were significantly different between healthy relatives of IBD patients and nonrelated healthy controls, between CD and UC patients in endoscopic remission, and between UC patients in relapse and non-UC individuals. Microbial load data combined with demographic and standard laboratory data improved the performance of the random forest models by 18%, reaching an average area under the receiver operating characteristic curve (AUC) of 0.842 (95% confidence interval [CI], 0.65 to 0.98), for IBD diagnosis and enhanced CD and UC discrimination and CD and UC relapse prediction. Our findings show that fecal fungal and bacterial loads could provide physicians with a noninvasive tool to discriminate disease subtypes or to predict disease flare in the clinical setting. IMPORTANCE Next-generation sequence data analysis has allowed a better understanding of the pathophysiology of IBD, relating microbiome composition and functions to the disease. Microbiome composition profiling may provide efficient diagnosis and prognosis tools in IBD. However, the bacterial and fungal loads of the fecal microbiota are underexplored as potential biomarkers of IBD. Ulcerative colitis (UC) patients have higher fecal fungal and bacterial loads than patients with ileal or ileocolonic CD. CD patients who relapsed harbor more-unstable fungal and bacterial loads than those of relapsed UC patients. Fecal fungal and bacterial load data improved prediction performance by 18% for IBD diagnosis based solely on clinical data and enhanced CD and UC discrimination and prediction of CD and UC relapse. Combined with existing laboratory biomarkers such as fecal calprotectin and C-reactive protein (CRP), microbial loads may improve the diagnostic accuracy of IBD and of ileal CD and UC disease activity and prediction of UC and ileal CD clinical relapse. This work was funded by Instituto de Salud Carlos III, grant PI17/00614, cofinanced by the European Regional Development Fund (ERDF) and by the PERIS (SLT002/16). F. Casellas has received research funding from AbbVie, Ferring, MSD, Shire, and Zambon and speaker fees from AbbVie, Chiesi, Ferring, Gebro, MSD, Shire, Takeda, and Zambon. S. Vermeire has received grant support from AbbVie, MSD, Pfizer, J&J, and Takeda; received speaker fees from AbbVie, MSD, Takeda, Ferring, Dr. Falk Pharma, Hospira, Pfizer Inc., and Tillots; and served as a consultant for AbbVie, MSD, Takeda, Ferring, Genentech/Roche, Robarts clinical trials, Gilead, Celgene, Prometheus, Avaxia, Prodigest, Shire, Pfizer Inc, Galapagos, Mundipharma, Hospira, Celgene, Second Genome, and Janssen. C. Manichanh has received financial support for research from Danone.

Background: The remission of Crohn's disease (CD) can be accomplished by faecal microbiota transplantation (FMT). However, this procedure has a low success rate, which could be attributed to mis-communication between recipient intestinal mucosa and donor microbiota. Methods: Here we used a human explant tissue model and an in vivo mouse model to examine changes in recipient intestinal mucosa upon contact with a faecal suspension (FS) obtained from a healthy donor. CD patients provided resected inflamed and non-inflamed mucosal tissues, whereas control colonic mucosa samples were collected from colorectal cancer patients. For the models, mucosal microbiome composition and tissue response were evaluated. Findings: We show that cytokine release and tissue damage were significantly greater in inflamed compared to non-inflamed CD tissues. Moreover, mucosal samples harbouring an initial low microbial load presented a shift in composition towards that of the FS, an increase in the relative count of Faecalibacterium prausnitzii, and a higher secretion of anti-inflammatory cytokine IL-10 compared to those with a high microbial load. Interpretation: Our results indicate that FMT during active inflammatory disease can compromise treatment outcome. We recommend the stratification of FMT recipients on the basis of tissue microbial load as a strategy to ensure successful colonization. Funding: This study was supported by grants from the Instituto de Salud Carlos III/FEDER (PI17/00614), the European Commission: (INCOMED-267128) and PERIS (SLT002/16). K.M. is a postdoctoral fellow and S.V. a senior clinical investigator of the Fund for Scientific Research Flanders, Belgium (FWO-Vlaanderen). Keywords: Intestinal microbiome, Crohn's disease, FMT, Recipient stratification, Microbial load, Anti-inflammatory cytokines

Cirrhosis severity in patients was previously shown to be associated with progressive changes in the fecal microbiome in a longitudinal setting. Recent evidence shows that bacterial translocation from the gut to the extraintestinal sites could play a major role in poor disease outcome and patient survival. However, the underlying mechanisms involving the microbiota in the disease progression are not well understood. Here, using an animal model of cirrhosis in a longitudinal and multibody sites setting, we showed the presence of a distinct composition of the microbiome in mesenteric lymph nodes, blood, and ascitic fluid compared to that in feces and ileocecal content, suggesting compartmentalization of the gut microbiome. We also demonstrate that microbiome changes in intestinal sites are associated with shifts in specific microbial groups in the mesenteric lymph nodes as well as an increase in systemic cytokine production, linking inflammation to decompensated cirrhosis in the gut-liver-immunity axis., Whether the interaction between the gut microbiota and the immune response influences the evolution of cirrhosis is poorly understood. We aimed to investigate modifications of the microbiome and the immune response during the progression of cirrhosis. Rats were treated with carbon tetrachloride (CCl4) to induce cirrhosis. We then assessed microbiome load and composition in stool, ileocecal contents (ICCs), mesenteric lymph nodes (MLNs), blood, and ascitic fluids (AFs) at 6, 8, and 10 weeks or ascites production and measured cytokine production in MLNs and blood. The microbiome of MLN, blood, and AF showed a distinct composition compared to that of stool and ICCs. Betaproteobacteria (Sutterella) were found associated with the appearance of a decompensated state of cirrhosis. Microbial load increased and showed a positive correlation with the relative abundance of pathobionts in the MLN of decompensated rats. Among several genera, Escherichia and “Candidatus Arthromitus” positively correlated with elevated levels of systemic proinflammatory cytokines. “Candidatus Arthromitus,” a segmented filamentous bacteria, was detected in ICC, MLN, and AF samples, suggesting a possible translocation from the gut to the AF through the lymphatic system, whereas Escherichia was detected in ICC, MLN, AF, and blood, suggesting a possible translocation from the gut to the AF through the bloodstream. In the present study, we demonstrate that microbiome changes in distinct intestinal sites are associated with microbial shifts in the MLNs as well as an increase in cytokine production, providing further evidence of the role the gut-liver-immunity axis plays in the progression of cirrhosis. IMPORTANCE Cirrhosis severity in patients was previously shown to be associated with progressive changes in the fecal microbiome in a longitudinal setting. Recent evidence shows that bacterial translocation from the gut to the extraintestinal sites could play a major role in poor disease outcome and patient survival. However, the underlying mechanisms involving the microbiota in the disease progression are not well understood. Here, using an animal model of cirrhosis in a longitudinal and multibody sites setting, we showed the presence of a distinct composition of the microbiome in mesenteric lymph nodes, blood, and ascitic fluid compared to that in feces and ileocecal content, suggesting compartmentalization of the gut microbiome. We also demonstrate that microbiome changes in intestinal sites are associated with shifts in specific microbial groups in the mesenteric lymph nodes as well as an increase in systemic cytokine production, linking inflammation to decompensated cirrhosis in the gut-liver-immunity axis.

Melanoma is a highly lethal cutaneous tumor, killing affected patients through development of multiple poorly immunogenic metastases. Suboptimal activation of immune system by melanoma cells is often due to molecular modifications occurring during tumor progression that prevent efficient recognition of melanoma cells by immune effectors. Statins are HMG-CoA reductase inhibitors, which block the mevalonate synthesis pathway, used by millions of people as hypocholesterolemic agents in cardiovascular and cerebrovascular diseases. They are also known to inhibit Rho GTPase activation and Rho dependent signaling pathways. Rho GTPases are regarded as molecular switches that regulate a wide spectrum of cellular functions and their dysfunction has been characterized in various oncogenic process notably in melanoma progression. Moreover, these molecules can modulate the immune response. Since 10 years we have demonstrated that Statins and other Rho GTPases inhibitors are critical regulators of molecules involved in adaptive and innate anti-melanoma immune response. In this review we summarize our major observations demonstrating that these pharmacological agents stimulate melanoma immunogenicity and suggest a potential use of these molecules to promote anti-melanoma immune response.

Inflammatory bowel disease; Faecal microbiota transplantation; Rat model of colitis Enfermedad inflamatoria intestinal; Trasplante de microbiota fecal; Modelo de colitis en ratas Malaltia inflamatòria intestinal; Trasplantament de microbiota fecal; Model de colitis en rates Background: Faecal microbiota transplantation (FMT) is a novel potential therapy for inflammatory bowel diseases, but it is poorly characterised. Methods: We evaluated the performance of the mouse and rat as a pre-clinical model for human microbiota engraftment. We then characterised the effect of a single human stool transfer (HST) on a humanised model of DSS-induced colitis. Colonic and faecal microbial communities were analysed using the 16S rRNA approach and clinical manifestations were assessed in a longitudinal setting. Findings: The microbial community of rats showed greater similarity to that of humans, while the microbiome of mice showed less similarity to that of humans. Moreover, rats captured more human microbial species than mice after a single HST. Using the rat model, we showed that HST compensated faecal dysbiosis by restoring alpha-diversity and by increasing the relative abundance of health-related microbial genera. To some extent, HST also modulated the microbial composition of colonic tissue. These faecal and colonic microbial communities alterations led to a relative restoration of colon length, and a significant decrease in both epithelium damage and disease severity. Remarkably, stopping inflammation by removing DSS before HST caused a faster and greater recovery of both microbiome and clinical manifestation features. Interpretation: Our results indicate that the rat outperforms the mouse as a model for human microbiota engraftment and show that the efficacy of HST can be enhanced when inflammation stimulation is withdrawn. Finally, our findings support a new therapeutic strategy based on the use FMT combined with anti inflammatory drugs. Study funded by the Instituto de Salud Carlos III/FEDER (PI17/00614), a government agency. The funder had no role in study design, data collection, data analysis, interpretation or writing of the report.

International audience; BACKGROUND & AIMS:Faecalibacterium prausnitzii, a member of the Clostridium IV group of the Firmicutes phylum that is abundant in the intestinal microbiota, has anti-inflammatory effects. The relative level of F prausnitzii is decreased in fecal samples from patients with inflammatory bowel diseases (IBDs) compared with healthy individuals. Reduced F prausnitzii was correlated with relapse of Crohn's disease after surgery. We identified, in human colonic mucosa and blood, a population of T regulatory type 1-like T regulatory (TREG) cells that express CD4 and CD8α (DP8α T cells) and are specific for F prausnitzii. We aimed to determine whether they are altered in patients with IBD.METHODS:We isolated DP8α T cells from human colon lamina propria and blood samples and used flow cytometry to detect markers of cells that are of colon origin. We quantified DP8α cells that express colon-specific markers in blood samples from 106 patients with IBD, 12 patients with infectious colitis, and 35 healthy donors (controls). We identified cells that respond to F prausnitzii. Cells were stimulated with anti-CD3, and their production of interleukin 10 was measured by enzyme-linked immunosorbent assay. We compared the frequency and reactivity of cells from patients vs controls using the 2-sided Student t test or 1-way analysis of variance.RESULTS:Circulating DP8α T cells that proliferate in response to F prausnitzii express the C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 6 (CXCR6). These cells also have features of TREG cells, including production of IL-10 and inhibition of T-cell proliferation via CD39 activity. The proportion of circulating CCR6+/CXCR6+ DP8α T cells was significantly reduced (P < .0001) within the total population of CD3+ T cells from patients with IBD compared with patients with infectious colitis or controls. A threshold of

Faecalibacterium prausnitzii, a member of the Clostridium IV group of the Firmicutes phylum that is abundant in the intestinal microbiota, has anti-inflammatory effects. The relative level of F prausnitzii is decreased in fecal samples from patients with inflammatory bowel diseases (IBDs) compared with healthy individuals. Reduced F prausnitzii was correlated with relapse of Crohn's disease after surgery. We identified, in human colonic mucosa and blood, a population of T regulatory type 1-like T regulatory (TWe isolated DP8α T cells from human colon lamina propria and blood samples and used flow cytometry to detect markers of cells that are of colon origin. We quantified DP8α cells that express colon-specific markers in blood samples from 106 patients with IBD, 12 patients with infectious colitis, and 35 healthy donors (controls). We identified cells that respond to F prausnitzii. Cells were stimulated with anti-CD3, and their production of interleukin 10 was measured by enzyme-linked immunosorbent assay. We compared the frequency and reactivity of cells from patients vs controls using the 2-sided Student t test or 1-way analysis of variance.Circulating DP8α T cells that proliferate in response to F prausnitzii express the C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 6 (CXCR6). These cells also have features of TWe identified a population of gut-derived T

The progression of cirrhosis is associated with alterations in the composition of the gut microbiome. To assess microbial translocation, we compared the serum microbial composition of patients with and without ascites and characterized the ascitic fluid microbiome using 16S rDNA high-throughput sequencing data. A complex and specific microbial community was detected in the serum and ascitic fluid of patients with cirrhosis but barely detectable in the serum of healthy controls. The serum microbiome of patients with ascites presented higher levels of lipopolysaccharide binding protein, a marker of microbial translocation, associated with higher diversity and relative abundance of Clostridiales and an unknown genus belonging to the Cyanobacteria phylum compared to patients without ascites. The composition of the fecal microbiome was also more altered in patients with than without ascites, confirming previous studies on fecal microbiome. We propose that alteration of the serum and fecal microbiome composition be considered indicators of cirrhosis progression.

Objective A decade of microbiome studies has linked IBD to an alteration in the gut microbial community of genetically predisposed subjects. However, existing profiles of gut microbiome dysbiosis in adult IBD patients are inconsistent among published studies, and did not allow the identification of microbial signatures for CD and UC. Here, we aimed to compare the faecal microbiome of CD with patients having UC and with non-IBD subjects in a longitudinal study. Design We analysed a cohort of 2045 non-IBD and IBD faecal samples from four countries (Spain, Belgium, the UK and Germany), applied a 16S rRNA sequencing approach and analysed a total dataset of 115 million sequences. Results In the Spanish cohort, dysbiosis was found significantly greater in patients with CD than with UC, as shown by a more reduced diversity, a less stable microbial community and eight microbial groups were proposed as a specific microbial signature for CD. Tested against the whole cohort, the signature achieved an overall sensitivity of 80% and a specificity of 94%, 94%, 89% and 91% for the detection of CD versus healthy controls, patients with anorexia, IBS and UC, respectively. Conclusions Although UC and CD share many epidemiologic, immunologic, therapeutic and clinical features, our results showed that they are two distinct subtypes of IBD at the microbiome level. For the first time, we are proposing microbiomarkers to discriminate between CD and non-CD independently of geographical regions.

CD70 is a costimulatory molecule member of the Tumor Necrosis Factor family that is expressed on activated immune cells. Its ectopic expression has been described in several types of cancer cells including lymphomas, renal cell carcinomas and glioblastomas. We have recently described its expression in a part of tumor cells from the vast majority of melanoma biopsies and human melanoma cell lines, and found that CD70 expression decreased over time as the disease progressed. Here, we show that RhoA, BRAF and Mitogen Activating Protein Kinase pathways are involved in the positive transcriptional regulation of CD70 expression in melanomas. Interestingly, the clinical inhibitor of the common BRAF V600E/D variants, Vemurafenib (PLX-4032), which is currently used to treat melanoma patients with BRAF V600E/D-mutated metastatic melanomas, decreased CD70 expression in human CD70+ melanoma cell lines. This decrease was seen in melanoma cells both with and without the BRAFV600E/D mutation, although was less efficient in those lacking the mutation. But interestingly, by silencing CD70 in CD70+ melanoma cell lines we show that PLX-4032-induced melanoma cell killing and its inhibitory effect on MAPK pathway activation are unaffected by CD70 expression. Consequently, our work demonstrates that CD70 ectopic expression in melanomas is not a valuable biomarker to predict tumor cells sensitivity to BRAF V600 inhibitors.

Background: CD70 is a costimulatory molecule of the tumour necrosis factor family expressed in activated immune cells and some solid tumours. In lymphocytes CD70 triggers T cell-mediated cytotoxicity and mitogen-activated protein kinase phosphorylation. Methods: We evaluated the expression of CD70 in biopsies and melanoma cell lines. Using melanoma cell lines positive or not for CD70, we analysed CD70 function on melanoma progression. Results: We report CD70 expression in human melanoma cell lines and tumour cells from melanoma biopsies. This expression was observed in 95% of primary melanomas but only 37% of metastases. Both monomeric and trimeric forms of CD70 were detected in tumour cell membrane fractions, whereas cytoplasmic fractions contained almost exclusively monomeric CD70. In vitro and in vivo experiments demonstrated that CD70 expression inhibited melanoma cell migration, invasion and pulmonary metastasis implantation independently of the tumour immune microenvironment. Increasing the levels of the trimeric form of CD70 through monoclonal antibody binding led to an increase in CD70+ melanoma cell invasiveness through MAPK pathway activation, RhoE overexpression, ROCK1 and MYPT1 phosphorylation decrease, and stress fibres and focal adhesions disappearance. Conclusions: Our results describe a new non-immunological function of melanoma-expressed CD70, which involves melanoma invasiveness through MAPK pathway, RhoE and cytoskeletal modulation.

The uptake and long-term cross-presentation of tumor Ag long peptides (LP) by dendritic cells (DC) make them attractive cancer vaccine candidates. However, it remains to be established whether LP can prime long-lived tumor-reactive CTL and whether other cell types are able to cross-present them. Using HLA-A2 healthy donor and melanoma patient-derived PBMC, we studied the in vitro cross-priming potential of Melan-A 16–40 LP bearing the HLA-A2–restricted epitope 26–35 or its analog 26–35A27L and compared it to the priming capacity of the short analog. We then addressed LP priming capacity in vivo using HLA-A2 mice. We also studied LP cross-presentation by monocyte-derived DC, plasmacytoid DC, monocytes, and B cells. We showed that the modified LP gave rise to high and sustained cross-presentation by monocyte-derived DC. This led to cross priming in vitro and in vivo and to expansion of long-lived tumor-reactive cytotoxic T cells. In contrast, the LP containing the natural 26–35 epitope primed specific T cells poorly, despite its long-lived cross-presentation, and T cells primed against the short analog were short-lived. We further showed that LP cross-presentation is restricted to monocytes and conventional DC. These results document for the first time, to our knowledge, the strong immunogenicity of a human tumor Ag LP. Of note, they underscore that this property is critically dependent on sufficient HLA binding affinity and/or TCR ligand potency of the cross-presented epitope. We conclude that LP fulfilling this requirement should be used as tumor vaccines, together with DC maturating agents, especially the Melan-A 16–40A27L LP, for the treatment of HLA-A2+ melanoma patients.

International audience; High level of T-cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of thisinfiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype andfunctional features of tumor-reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated fromCRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivityof CRC TIL and tumor-reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions(up to 18%) of double positive CD41CD8ab1 (DP) ab T cells. Interestingly, a high proportion (16–20%) of this TIL subsetdisplayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in mostCRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DPTIL were polyclonal, restricted by HLA class-I, proliferated poorly and secreted higher amounts of IL-4 and IL-13 than singlepositive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increasedfrequencies of tumor-reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of thiscancer. Based on their high secretion of IL-4 and IL-13 and on previously described roles of these cytokines in cancers, wepostulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.

International audience; Signaling via interleukin (IL)-10 or transforming growth factor (TGF)-β is disrupted in subpopulations of patients with inflammatory bowel disease, but it is not clear how a T-helper (Th) 1 cell response is induced. We studied conversion of human mucosal innate immune cells into inflammatory cells and the initiation of a Th1 cell response following loss of IL-10 or TGF-β signaling.METHODS:We depleted IL-10 or TGF-β from explant cultures of human normal colonic mucosa using immunoneutralization. Pharmacologic inhibitors and antibodies were used to determine the factors involved in the initiation of an interferon (IFN)-γ response following loss of TGF-β or IL-10 signaling. Cytokines produced by mucosal cells were assessed by enzyme-linked immunosorbent assay and quantitative reverse-transcriptase polymerase chain reaction. The subsets of cells involved in cytokine production were determined by in situ immunofluorescence analysis and flow cytometry after digestion of the explants with collagenase.RESULTS:Depletion of IL-10 from human normal colonic mucosa resulted in an IFN-γ response, characterized by early-stage secretion of mature IL-18 and production of the active form of caspase-1 by macrophages and some epithelial cells. A caspase-1 inhibitor or the IL-18 antagonist IL-18-binding protein blocked this response. By contrast, depletion of TGF-β resulted in an IFN-γ response that was preceded by and required secretion of IL-12 from macrophages, dendritic cells, and epithelial cells.CONCLUSIONS:Innate immune cells (macrophages and epithelial cells) activate a Th1 cell response in explant cultures of human normal colonic mucosa depleted in IL-10 or TGF-β via distinct, nonredundant pathways. These pathways might contribute to the pathogenesis of inflammatory bowel disease.

International audience; BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-gamma; and iv) anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

The capacity of FasL molecules expressed on melanoma cells to induce lymphocyte apoptosis contributes to either antitumor immune response or escape depending on their expression level. Little is known, however, about the mechanisms regulating FasL protein expression. Using the murine B16F10 melanoma model weakly positive for FasL, we demonstrated that in vitro treatment with statins, inhibitors of 3-hydroxy3-methylgutaryl CoA reductase, enhances membrane FasL expression. C3 exotoxin and the geranylgeranyl transferase I inhibitor GGTI-298, but not the farnesyl transferase inhibitor FTI-277, mimic this effect. The capacity of GGTI-298 and C3 exotoxin to inhibit RhoA activity prompted us to investigate the implication of RhoA in FasL expression. Inhibition of RhoA expression by small interfering RNA (siRNA) increased membrane FasL expression, whereas overexpression of constitutively active RhoA following transfection of RhoAV14 plasmid decreased it. Moreover, the inhibition of a RhoA downstream effector p160ROCK also induced this FasL overexpression. We conclude that the RhoA/ROCK pathway negatively regulates membrane FasL expression in these melanoma cells. Furthermore, we have shown that B16F10 cells, through the RhoA/ROCK pathway, promote in vitro apoptosis of Fas-sensitive A20 lymphoma cells. Our results suggest that RhoA/ROCK inhibition could be an interesting target to control FasL expression and lymphocyte apoptosis induced by melanoma cells.

Defective antitumor immune responses are frequent consequences of defects in the expression of major histocompatibility complex (MHC) class I and costimulatory molecules. We demonstrated that statins, inhibitors of HMGCoA reductase, enhance mIFN-gamma induced expression of MHC class I antigens on murine B16F10 melanoma. GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor, mimics this effect of statins. This effect is related to peptide transporter protein TAP1 up-regulation. Simultaneously, GGTI-298 induces the expression of CD80 and CD86 costimulatory molecules. C3 exoenzyme, which selectively inactivates Rho proteins, phenocopies the effects of GGTI-298, indicating a role for Rho proteins in these events. Furthermore, the treatment of B16F10 cells with GGTI-298 or C3 exoenzyme associated with mIFN-gamma induces in vivo tumor growth slowing down in immunocompetent but not in nu/nu syngeneic mice. Both in vivo injections and in vitro restimulation of splenocytes with GGTI-298- and mIFN-gamma-treated B16F10 cells induces an enhancement of specific CD8 T lymphocytes labeled by TRP-2/H-2K(b) tetramers. Finally, these effects are not limited to mouse models since they were also reproduced in two human melanoma cell lines. These observations indicate that protein geranylgeranylation as well as Rho protein are critical for costimulatory and IFN-gamma-dependent MHC class I molecule expression in melanoma.

INTRODUCTION: We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta. METHODS: The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha. The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity. RESULTS: FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established. CONCLUSIONS: Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.