Erythropoietin induces tumour progression and CD39 expression on immune cells in a preclinical model of triple-negative breast cancer.

The adverse effects observed in some cancer patients treated with erythropoiesis-stimulating agents such as erythropoietin (EPO) might be due to the latter's well-known immunosuppressive functions. Here, we used a mouse model of syngeneic triple-negative breast cancer to explore EPO's immunomodulatory role in a tumour setting. Our results showed that EPO treatment promotes tumour growth, exacerbates the 'immune desert', and results in a 'cold tumour'. EPO treatment changed the immune cell distribution in peripheral blood, secondary lymphoid organs, and the tumour microenvironment (TME). Our in-depth analysis showed that EPO mainly impacts CD4 T cells by accelerating their activation in the spleen and thus their subsequent exhaustion in the TME. This process is accompanied by a general elevation of CD39 expression by several immune cells (notably CD4 T cells in the tumour and spleen), which promotes an immunosuppressive TME. Lastly, we identified a highly immunosuppressive CD39+ regulatory T cell population (ICOS+, CTLA4+, Ki67+) as a potential biomarker of the risk of EPO-induced tumour progression. EPO displays pleiotropic immunosuppressive functions and enhances mammary tumour progression in mice.

Gut Microbiota Composition in Long-Remission Ulcerative Colitis is Close to a Healthy Gut Microbiota.

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 < .001). Logistic regression analysis showed that a high Shannon index (odds ratio, 4.83; 95% confidence interval, 1.5-20.6) or presence of A. muciniphila (odds ratio, 4.9; 95% confidence interval, 1.12-29.08) in fecal samples at entry was independently associated with clinical remission over a follow-up period of 24 months. 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.

Microbiome studies have shown low gut microbial richness and diversity in ulcerative colitis patients. Patients who achieve long-term remission show evidence of substantial recovery of the gut microbial ecosystem in terms of diversity and composition.

Spheroplexes: Hybrid PLGA-cationic lipid nanoparticles, for in vitro and oral delivery of siRNA.

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.

Advances in CAR-T Cell Genetic Engineering Strategies to Overcome Hurdles in Solid Tumors Treatment.

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.

Mucosal microbial load in Crohn's disease: A potential predictor of response to faecal microbiota transplantation.

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).

A single faecal microbiota transplantation modulates the microbiome and improves clinical manifestations in a rat model of colitis.

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.

Sequential Changes in the Mesenteric Lymph Node Microbiome and Immune Response during Cirrhosis Induction in Rats.

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.

Faecalibacterium prausnitzii Skews Human DC to Prime IL10-Producing T Cells Through TLR2/6/JNK Signaling and IL-10, IL-27, CD39, and IDO-1 Induction.

The human colonic mucosa contains regulatory type 1-like (Tr1-like, i.e., IL-10-secreting and Foxp3-negative) T cells specific for the gut Clostridium Faecalibacterium prausnitzii (F. prausnitzii), which are both decreased in Crohn's disease patients. These data, together with the demonstration, in mice, that colonic regulatory T cells (Treg) induced by Clostridium bacteria are key players in colon homeostasis, support a similar role for F. prausnitzii-specific Treg in the human colon. Here we assessed the mechanisms whereby F. prausnitzii induces human colonic Treg. We demonstrated that F. prausnitzii, but not related Clostridia, skewed human dendritic cells to prime IL-10-secreting T cells. Accordingly, F. prausnitzii induced dendritic cells to express a unique array of potent Tr1/Treg polarizing molecules: IL-10, IL-27, CD39, IDO-1, and PDL-1 and, following TLR4 stimulation, inhibited their up-regulation of costimulation molecules as well as their production of pro-inflammatory cytokines IL-12 (p35 and p40) and TNFα. We further showed that these potent tolerogenic effects relied on F. prausnitzii-induced TLR2/6 triggering, JNK signaling and CD39 ectonucleotidase activity, which was induced by IDO-1 and IL-27. These data, together with the presence of F. prausnitzii-specific Tr1-like Treg in the human colon, point out to dendritic cells polarization by F. prausnitzii as the first described cellular mechanism whereby the microbiota composition may affect human colon homeostasis. Identification of F. prausnitzii-induced mediators involved in Tr1-like Treg induction by dendritic cells opens therapeutic avenues for the treatment of inflammatory bowel diseases.

Expression of CCR6 and CXCR6 by Gut-Derived CD4+/CD8α+ T-Regulatory Cells, Which Are Decreased in Blood Samples From Patients With Inflammatory Bowel Diseases.

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 <7.875 CCR6+/CXCR6+ DP8α T cells/10,000 CD3+ cells discriminated patients with IBD from those with infectious colitis with 100% specificity and 72.2% sensitivity. CONCLUSIONS: We identified a population of gut-derived TREG cells that are reduced in blood samples from patients with IBD compared with patients with infectious colitis or controls. These cells should be studied further to determine the mechanisms of this reduction and how it might contribute to the pathogenesis of IBD and their prognostic or diagnostic value.

A microbial signature for Crohn's disease.

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.

Regulatory properties of statins and rho gtpases prenylation inhibitiors to stimulate melanoma immunogenicity and promote anti-melanoma immune response.

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.

Alteration of the serum microbiome composition in cirrhotic patients with ascites.

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.

Melanoma Expressed-CD70 Is Regulated by RhoA and MAPK Pathways without Affecting Vemurafenib Treatment Activity.

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.

Melanoma-expressed CD70 is involved in invasion and metastasis.

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.

Microbiota-Specific CD4CD8αα Tregs: Role in Intestinal Immune Homeostasis and Implications for IBD.

In studies in murine models, active suppression by IL-10-secreting Foxp3 regulatory T cells (Tregs) has emerged as an essential mechanism in colon homeostasis. However, the role of the equivalent subset in humans remains unclear, leading to suggestions that other subsets and/or mechanisms may substitute for Foxp3 Tregs in the maintenance of colon homeostasis. We recently described a new subset of CD4CD8αα T cells reactive to the gut bacterium Faecalibacterium prausnitzii and endowed with regulatory/suppressive functions. This subset is abundant in the healthy colonic mucosa, but less common in that of patients with inflammatory bowel disease (IBD). We discuss here the physiological significance and potential role of these Tregs in preventing inflammation of the gut mucosa and the potential applications of these discoveries for IBD management.

CD4CD8αα lymphocytes, a novel human regulatory T cell subset induced by colonic bacteria and deficient in patients with inflammatory bowel disease.

How the microbiota affects health and disease is a crucial question. In mice, gut Clostridium bacteria are potent inducers of colonic interleukin (IL)-10-producing Foxp3 regulatory T cells (Treg), which play key roles in the prevention of colitis and in systemic immunity. In humans, although gut microbiota dysbiosis is associated with immune disorders, the underlying mechanism remains unknown. In contrast with mice, the contribution of Foxp3 Treg in colitis prevention has been questioned, suggesting that other compensatory regulatory cells or mechanisms may exist. Here we addressed the regulatory role of the CD4CD8 T cells whose presence had been reported in the intestinal mucosa and blood. Using colonic lamina propria lymphocytes (LPL) and peripheral blood lymphocytes (PBL) from healthy individuals, and those with colon cancer and irritable bowel disease (IBD), we demonstrated that CD4CD8αα (DP8α) T lymphocytes expressed most of the regulatory markers and functions of Foxp3 Treg and secreted IL-10. Strikingly, DP8α LPL and PBL exhibited a highly skewed repertoire toward the recognition of Faecalibacterium prausnitzii, a major Clostridium species of the human gut microbiota, which is decreased in patients with IBD. Furthermore, the frequencies of DP8α PBL and colonic LPL were lower in patients with IBD than in healthy donors and in the healthy mucosa of patients with colon cancer, respectively. Moreover, PBL and LPL from most patients with active IBD failed to respond to F. prausnitzii in contrast to PBL and LPL from patients in remission and/or healthy donors. These data (i) uncover a Clostridium-specific IL-10-secreting Treg subset present in the human colonic LP and blood, (ii) identify F. prausnitzii as a major inducer of these Treg, (iii) argue that these cells contribute to the control or prevention of colitis, opening new diagnostic and therapeutic strategies for IBD, and (iv) provide new tools to address the systemic impact of both these Treg and the intestinal microbiota on the human immune homeostasis.

HLA anchor optimization of the melan-A-HLA-A2 epitope within a long peptide is required for efficient cross-priming of human tumor-reactive T cells.

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 26-35(A27L) 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 16-40(A27L) LP, for the treatment of HLA-A2(+) melanoma patients.

Loss of interleukin-10 or transforming growth factor ß signaling in the human colon initiates a T-helper 1 response via distinct pathways.

BACKGROUND & AIMS: Signaling via interleukin (IL)-10 or transforming growth factor (TGF)-ß 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-ß signaling. METHODS: We depleted IL-10 or TGF-ß 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-ß 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-ß 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-ß via distinct, nonredundant pathways. These pathways might contribute to the pathogenesis of inflammatory bowel disease.

Tumor-reactive CD4+ CD8αß+ CD103+ αßT cells: a prevalent tumor-reactive T-cell subset in metastatic colorectal cancers.

High level of T-cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor-reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of 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 CD4(+) CD8αß(+) (DP) αß T cells. Interestingly, a high proportion (16-20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class-I, proliferated poorly and secreted higher amounts of IL-4 and IL-13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor-reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL-4 and IL-13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.

Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

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.