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Supplementary Figure S6. Early cytokine production by APC in the vaccine draining lymph nodes

Supplementary Figure S2. Activation status of antigen-specific T cells following vaccination with and without adjuvants

Supplementary Figure S4. Draining lymph node T cell responses following vaccination of tumor-bearing mice

Supplementary Figure S1. T cell responses following peptide vaccination in combination with CpG

Supplementary Figure S5. Cytokine production by antigen-specific T cells at the peak of the effector response

Supplementary Figure S3. T cell responses following adjuvanted peptide vaccination with or without IFA

Dendritic cells (DCs) are professional antigen-presenting cells, which are optimal for the priming of a T cell response against pathogens and tumors. Therefore, many efforts are made to develop therapeutic cancer vaccines which preferentially target the antigen to DC subsets. To this aim, we developed two types of recombinant fusion proteins, which favor antigen delivery to pro-inflammatory DCs as well as the crosstalk between specialized subpopulations of DCs. The first approach combines peptide/CpG vaccination with the recruitment of iNKT cells to the tumor site via CD1d-antitumor scFv fusion proteins. The second approach is targeting the tumor antigen to cross-presenting Xcr1

Dendritic cells (DCs) are professional antigen-presenting cells, which are optimal for the priming of a T cell response against pathogens and tumors. Therefore, many efforts are made to develop therapeutic cancer vaccines which preferentially target the antigen to DC subsets. To this aim, we developed two types of recombinant fusion proteins, which favor antigen delivery to pro-inflammatory DCs as well as the crosstalk between specialized subpopulations of DCs. The first approach combines peptide/CpG vaccination with the recruitment of iNKT cells to the tumor site via CD1d-antitumor scFv fusion proteins. The second approach is targeting the tumor antigen to cross-presenting Xcr1+ DCs via a fusion protein made of Xcl1 fused to a synthetic long peptide followed by an IgG1 Fc fragment. Both strategies allow a potent tumor-specific CD8 T cell response associated with tumor regression or tumor growth delay depending on the model. In the case of iNKT cell activation, the strategy relies on a strong IL-12 release by splenic DCs, while in the second case, the T cell response is strictly dependent on the presence of Xcr1+ cross-presenting DCs.

BACKGROUND: Esophageal and gastroesophageal junctional (GEJ) adenocarcinoma is one of the most fatal cancers and has the fastest rising incidence rate of all cancers. Identification of biomarkers is needed to tailor treatments to each patient's tumor biology and prognosis. METHODS: Gene expression profiling was performed in a test cohort of 80 chemoradiotherapy (CRTx) naïve patients with external validation in a separate cohort of 62 CRTx naïve patients and 169 patients with advanced stage disease treated with CRTx. RESULTS: As a novel prognostic biomarker after external validation CD151 showed promise. Patients exhibiting high levels of CD151 (=median) had a longer median overall survival than patients with low CD151 tumor levels (median not reached vs. 30.9 months; p = 0.01). This effect persisted in a multivariable Cox regression model with adjustment for tumor stage [adjusted hazard ratio (aHR) 0.33; 95 confidence interval (CI) 0.14 0.78; p = 0.01] and was further corroborated through immunohistochemical analysis (aHR 0.22; 95 CI 0.08 0.59; p = 0.003). This effect was not found in the separate cohort of CRTx exposed patients. CONCLUSION: Tumoral expression levels of CD151 may provide independent prognostic information not gained by conventional staging of patients with esophageal and GEJ adenocarcinoma treated by esophagectomy alone.

The contribution of immune cells to the inflammasome that characterises type 2 diabetes mellitus and obesity is under intense research scrutiny. We hypothesised that early changes in glucose metabolism following gastric banding surgery may relate to systemic inflammation, particularly cell-mediated immunity.Obese participants (BMI 43.4 ± 4.9 kg/m(2), n = 15) with diabetes or impaired glucose tolerance (IGT) underwent laparoscopic adjustable gastric banding surgery. Measurements taken before, and at 2 and 12 weeks after surgery included: fasting glucose, glucose levels 2 h after a 75 g oral load, glucose incremental AUC, oral glucose insulin sensitivity index (OGIS), circulating immune cell numbers and activation, and adipokine levels. Subcutaneous and visceral adipose tissue were collected at surgery, and macrophage number and activation measured.There were significant reductions in fasting and 2 h glucose, as well as improved OGIS at 2 and 12 weeks. At 12 weeks, 80% of the diabetic participants reverted to normal glucose tolerance or IGT, and all IGT participants had normalised glucose tolerance. The 12 week fall in fasting glucose was significantly related to baseline lymphocyte and T lymphocyte numbers, and to granulocyte activation, but also to the magnitude of the 12 week reduction in lymphocyte and T lymphocyte numbers and TNF-α levels. In a model that explained 75% of the variance in the change in fasting glucose, the 12 week change in T lymphocytes was independently associated with the 12 week fall in fasting glucose.Rapid improvements in glucose metabolism after gastric banding surgery are related to reductions in circulating pro-inflammatory immune cells, specifically T lymphocytes. The contribution of immune cell-mediated inflammation to glucose homeostasis in type 2 diabetes and its improvement after bariatric surgery require further investigation.

Antitumor immunity is strongly influenced by the balance of tumor antigen-specific effector T cells (Teff) and regulatory T cells (Treg). However, the impact that vaccine adjuvants have in regulating the balance of antigen-specific T-cell populations is not well understood. We found that antigen-specific Tregs were induced following subcutaneous vaccination with either OVA or melanoma-derived peptides, with a restricted expansion of Teffs. Addition of the adjuvants CpG-ODN or Poly(I:C) preferentially amplified Teffs over Tregs, dramatically increasing the antigen-specific Teff:Treg ratios and inducing polyfunctional effector cells. In contrast, two other adjuvants, imiquimod and Quil A saponin, favored an expansion of antigen-specific Tregs and failed to increase Teff:Treg ratios. Following therapeutic vaccination of tumor-bearing mice, high ratios of tumor-specific Teffs:Tregs in draining lymph nodes were associated with enhanced CD8+ T-cell infiltration at the tumor site and a durable rejection of tumors. Vaccine formulations of peptide+CpG-ODN or Poly(I:C) induced selective production of proinflammatory type I cytokines early after vaccination. This environment promoted CD8+ and CD4+ Teff expansion over that of antigen-specific Tregs, tipping the Teff to Treg balance to favor effector cells. Our findings advance understanding of the influence of different adjuvants on T-cell populations, facilitating the rational design of more effective cancer vaccines. Cancer Res; 73(22); 6597–608. ©2013 AACR.

The origin of smooth muscle cells in developing atherosclerotic lesions is a controversial topic with accumulating evidence indicating that at least some arterial smooth muscle cells might originate from bone marrow-derived smooth muscle cell precursors circulating in the blood. The stem cell markers currently used for the identification of stem cells in the arterial intima can be expressed by a number of different cell types residing in the arterial wall, such as mast cells, endothelial cells and dendritic cells, which can make interpretation of the data obtained somewhat ambiguous. In the present study we examined whether the putative intestinal stem cell marker Musashi-1 is expressed in the arterial wall. Using a multiplexed tandem polymerase chain reaction method (MT-PCR) and immunohistochemistry, Musashi-1 expression was revealed in human coronary arterial wall tissue segments, and this finding was followed by the demonstration of significantly higher expression levels of Musashi-1 in atherosclerotic plaques compared with those in undiseased intimal sites. Double immunohistochemistry demonstrated that in the arterial wall Musashi-1 positive cells either did not display any specific markers of cells that are known to reside in the arterial intima or Musashi-1 was co-expressed by smooth muscle α-actin positive cells. Some Musashi-1 positive cells were found along the luminal surface of arteries as well as within microvessels formed in atherosclerotic plaques by neovascularization, which supports the possibility that Musashi-1 positive cells might intrude into the arterial wall from the blood and might even represent circulating smooth muscle cell precursors.

Type 2 diabetes mellitus (T2D) is predicted by central obesity and circulating adipokines regulating inflammation. We hypothesized that visceral adipose tissue (VAT) in T2D expresses greater levels of proinflammatory molecules. Paired samples of subcutaneous (SAT) and VAT were excised at elective surgery (n = 16, 6 with T2D, n = 8 age- and gender- matched controls). Metabolic parameters were measured in the fasted state: body composition by dual-energy X-ray absorptiometry and insulin action by hyperinsulinemic-euglycemic clamp. Adipose tissue mRNA gene expression was measured by quantitative reverse transcriptase-PCR. Subjects with T2D had higher VAT expression of molecules regulating inflammation (tumor necrosis factor-alpha (TNFalpha), macrophage inflammatory protein (MIP), interleukin-8 (IL-8)). Fasting glucose related to VAT expression of TNFalpha, MIP, serum amyloid A (SAA), IL-1alpha, IL-1beta, IL-8, and IL-8 receptor. Abdominal fat mass was related to VAT expression of MIP, SAA, cAMP response element-binding protein (CREBP), IL-1beta, and IL-8. Insulin action related inversely to VAT complement C3 expression only. There were depot-specific differences in expression of serum T2D predictors: VAT expressed higher levels of complement C3; SAT expressed higher levels of retinol-binding protein-4 (RBP4), adiponectin, and leptin. In summary, VAT in T2D expresses higher levels of adipokines involved in inflammation. VAT expression of these molecules is related to fasting glucose and insulin action. Increased production of these proinflammatory molecules by VAT may explain the links observed between visceral obesity, insulin resistance, and diabetes risk.

The cancer stem cell theory states that cancers contain tumor-forming cells that have the ability to self-renew as well as give rise to cells that differentiate. Cancer stem cells have been identified in several solid tumors, but stem cells in normal human esophagus or in Barrett's esophagus or adenocarcinoma have not been reported. Musashi-1 is expressed by the crypt base columnar cells identified as intestinal stem cells. In other diseases of the gastrointestinal tract, local inflammation of the tunica mucosa may be an initiating factor of alteration of focal tissue 'niches,' where dormant stem cells locate. The present study investigated whether Musashi-1 is expressed in the esophagus and its relation to immune inflammation of the mucosa in Barrett's esophagus and esophageal adenocarcinoma. A total of 41 esophageal tissue specimens from 41 patients were studied. Of these, 15 were esophageal adenocarcinoma, 17 were Barrett's esophagus (10 intestinal metaplasia and 7 dysplasia), and 9 were normal squamous esophagus tissue specimens from patients without esophageal pathology. Immunohistochemistry was performed using antibodies to Musashi-1 and to a set of cell type-specific markers. A multiplexed tandem polymerase chain reaction method was used to measure the relative mRNA expression levels of Musashi-1 and the specific dendritic cell marker dendritic cell-specific intercellular molecule-3 (ICAM-3)-grabbing nonintegrin. Immunohistochemistry demonstrated the presence of small numbers of Musashi-1+ cells scattered in the connective tissue stroma and within the epithelium in cardiac-type glands in biopsies from patients without Barrett's esophagus. Musashi-1 expression was present in Barrett's intestinal metaplasia and in dysplastic Barrett's in which the majority of epithelial cells in individual glands expressed this antigen. Expression of Musashi-1 was highest in esophageal adenocarcinoma, where it was most intense in glands that displayed features of early stages of adenocarcinoma formation. In contrast, Musashi-1 staining level was weaker in glands that displayed features of advanced adenocarcinoma. Double immunostaining with proliferating cell nuclear antigen showed low proliferation in the vast majority of Musashi-1+ cells. Musashi-1 mRNA expression levels were significantly higher in esophageal adenocarcinoma than in normal esophagus or Barrett's esophagus tissues. Dendritic cell-specific intercellular molecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) mRNA expression levels were significantly increased in both Barrett's tissues and adenocarcinoma tissues. Expression of the putative stem cell marker Musashi-1 is absent in normal squamous epithelium, weak in esophageal cardiac-type glands and Barrett's esophagus, and markedly increased in adenocarcinoma, especially in glands displaying features of early cancer development. Musashi-1 expressing cells may be significant in the etiology of Barrett's esophagus and adenocarcinoma, and perhaps even a cell of origin for this disease. We speculate that immune inflammation occurring in Barrett's esophagus alters the mucosal microenvironment in a manner which is favorable to the activation of dormant stem cells.

[Abstract] Different vaccine and adjuvant combinations are known to rapidly induce antigen presenting cell (APC) maturation and pro-inflammatory cytokine and production, which in turn play an important role in the priming of antigen-specific T cells. Measuring cytokine production systemically in the serum fails to detect localized responses in the lymph nodes draining a subcutaneous immunization site. On the other hand, stimulating APC with vaccine formulations in vitro lacks the complexity of the lymph node microenvironment and the presence of other in vivo factors. Here we analyse cytokine production directly in vaccine draining lymph nodes (dLN) extracted early after in vivo vaccination. To do this we perform cytokine multiplex analysis of supernatants from whole dLN cell suspensions following a brief ex vivo incubation.

[Abstract] Measuring antigen-specific T cell responses in the blood and lymphoid organs of vaccinated mice can give us a useful indication of the potency of a vaccine formulation. Unfortunately, systemic or even localized lymphoid T cell responses are not always predictive of the ability of a vaccine to induce tumor protection. Measuring the antigen-specific T cell response within the tumor infiltrating lymphocytes is a more accurate indicator or vaccine efficacy. However, multi-parameter flow cytometric analysis of T cells isolated from tumor tissue can be quite challenging due to the over-whelming number of tumor cells present in relation to the tumor infiltrating lymphocytes (TIL) and to problems associated to the large and adhesive nature of many tumor cell. Here we take advantage of a pre-flow separation of CD45 + leukocytes from the tumor tissue using the MACS magnetic cell sorting system, resulting in a much cleaner cell preparation with which to proceed to flow cytometric staining and analysis.

Weight loss after bariatric surgery reduces cardiac risk and morbidity. We examined weight loss effects on arterial stiffness in morbidly obese subjects, in relation to cytokines, circulating and subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT)-based immune cells and gene expression. Obese subjects with type 2 diabetes mellitus (T2D) or impaired glucose tolerance ( n = 14, mean ± SEM body mass index 42.9 kg/m2) underwent 24 weeks’ caloric restriction, with gastric banding at 12 weeks. Measures were: arterial augmentation index (AIx), insulin resistance, circulating cytokines, immune cell activation markers, and SAT and VAT cytokine gene expression. Weight loss reduced AIx by 20% ( p = 0.007), with falls in s-selectin ( p = 0.001) and inter-cellular adhesion molecule ( p = 0.04). Improved AIx related to reduced surface expression of the interleukin (IL)-2 receptor on T-lymphocytes (TL-IL2R) and granulocyte adhesion markers ( r = 0.59, 0.64, respectively, p < 0.04). Higher VAT expression of interferon-γ and monocyte chemoattractant protein-1 associated with a blunted AIx response. A model of TL-IL2R expression, waist, weight and insulin resistance explained 73% of the variance in AIx reduction ( p = 0.005). In morbidly obese dysglycaemic subjects, modest weight loss reduces arterial stiffness, the magnitude of which relates to improved markers of inflammation.

Widespread applicability of tissue-based mRNA expression screening for Barrett esophagus (BE) is likely to require (1) accurate methods for assaying archival formalin-fixed, paraffin-embedded (FFPE) histopathology specimens taken at endoscopy, and (2) validation studies of promising biomarkers in different patient cohorts.30 genes were significantly differentially expressed by histopathology tissue type. The direction and magnitude of the differences were very similar to those found in previous studies using fresh frozen tissues. Novel upregulated genes were TSPAN8 (also known as CO-029), TSPAN24 (CD151), EGR1 and TCIRG1. Novel downregulated genes were DPYD, TSPAN29 (CD9) and Ets1. Strong associations between histopathology type and gene expression were observed with the overexpressed genes: cyclo-oxygenase-2 (COX-2), for which histopathology type explained 77.7% of the variation in expression, TSPAN1 (73.5%), TSPAN8 (62.9%), SPARC (62.1%), MMP7 (50.8%); and the under-expressed genes ADH7 (53.7%), APC (58.2%), RAR-gamma (51.3%).mRNA was isolated from 54 FFPE small endoscopic biopsies from patients with Barrett intestinal metaplasia (BE), esophageal adenocarcinoma (EAC), or control patients with a normal squamous-lined esophagus. Multiplexed tandem PCR (MT-PCR) was used to quantitate 50 selected candidate genes for BE and control genes in duplicate. Principal component analysis (PCA) was conducted to explore the presence of global differences in gene expression profiles. One-way analysis of variance (ANOVA) of the transformed data was used to identify genes that were differentially expressed between different histological subtypes. Differentially expressed genes with a fold change of ≥2 (upregulated) or ≤-2 (downregulated) are reported with the p value for each comparison (EAC vs. normal, EAC vs. BE and BE vs. normal). The Benjamini-Hochberg method was used to control the false discovery rate at 0.01 for all comparisons.Alterations in expression of select genes are strongly associated with BE or EAC or both. This study's findings for many highly differentially expressed genes are very similar to those previously reported, suggesting that these genes should be tested further in longitudinal studies for their potential role as biomarkers of progression to more advanced Barrett disease.

The thymidine kinase gene of the herpes simplex virus (HSV-tk) is a suicide gene when administrated with the prodrug ganciclovir (GCV). This study investigated the effectiveness of HSV-tk activation as gene therapy for gastroesophageal junction and gastric adenocarcinomas using either the stress-inducible Grp78 promoter or the murine leukemia virus long-terminal repeat (LTR) promoter.The HSV-tk gene, controlled by either the Grp78 promoter or the LTR promoter, was transduced into the gastroesophageal junction adenocarcinoma cell line SK-GT-5 and the gastric adenocarcinoma cell line MKN-74. Cell viability after exposure to varying concentrations of GCV was compared. The same cell lines were used to develop a nude mouse model for studies of the HSV-tk/GCV effect in vivo. The effect of intraperitoneal GCV injection on growth of the subcutaneous tumors was measured. HSV-TK expression was measured by Western blot and reverse transcription polymerase chain reaction.Cell viability in vitro was significantly lower in the HSV-tk expressing (HSV-tk+) cells compared to control (no HSV-tk) cells after exposure to GCV. MKN-74tk+ cells were more sensitive to GCV killing than SK-GT-5tk+ cells. After culture with 1 microg/ml GCV for 10 days, MKN-74/tk cells were totally killed, whereas most SK-GT-5/tk cells survived. Cell viability was significantly lower under glucose starvation conditions when HSV-tk expression was regulated by the Grp78 promoter compared with the LTR promoter. MKN-74 tumors formed with HSV-tk+ cells in nude mice were eliminated after administration of GCV for 3 weeks, but GCV had no effect on tumors formed from HSV-tk- cells. Eradication of tumor formed with Grp78-tk cells was faster than that with LTR-tk cells. HSV-TK protein and mRNA were expressed in the transduced, but not the non-transduced tumors.HSV-tk xwith ganciclovir suicide gene therapy results in significant cell killing in gastroesophageal junction and gastric adenocarcinoma cells both in vitro and in vivo, but complete tumor elimination only occurred with the gastric adenocarcinoma cell tumors. The most effective approach in this study used the Grp78 promoter in glucose-starvation stress conditions.

FTO gene variants are linked to obesity. We tested for site-specific differences in FTO gene expression in subcutaneous and visceral adipose tissue (SAT and VAT, respectively) from individuals with and without type 2 diabetes mellitus (T2D) and the relationships between fasting glucose, in vivo insulin action, and measures of adiposity with FTO gene expression in adipose tissue.Paired subcutaneous and visceral fat were excised at elective surgery in n = 16 subjects (six with T2D, age-matched). Metabolic parameters were measured in fasted state; body composition by dual-energy X-ray absorptiometry; and insulin action by hyperinsulinemic euglycemic clamp. Adipose tissue mRNA gene expression was determined by quantitative RT-PCR.Subjects with T2D had SAT and VAT FTO mRNA expression similar to controls. There was no depot specificity between SAT and VAT FTO mRNA expression. Insulin action did not relate to SAT or VAT FTO mRNA expression. SAT FTO mRNA expression was related to fasting glucose and waist circumference only. SAT and VAT FTO mRNA expression was not related to direct measures of total or central abdominal adiposity. SAT FTO mRNA expression was related to SAT tumor necrosis factor-alpha and nuclear factor-kappaB mRNA expression.FTO gene expression is not increased in SAT and VAT in T2D and does not relate to insulin action. The links between FTO and metabolic complications of diabetes require further elucidation.

Background: Endoscopic treatment by either radiofrequency (RFA) ablation or endoscopic mucosal resection (EMR) has become first line treatment for Barrett's esophagus (BE) with high grade dysplasia, while focal EMR and subsequent elimination of the residual segment by RFA or EMR is accepted therapy for intramucosal cancer (IMC). RFA and EMR are known to be clinically effective but the effect on the mRNA expression of molecular markers of disease stage, which may inform the long term risk of progression to adenocarcinoma, is unknown. We compared the mRNA expression profile in pre-treatment BE tissues biopsies and post-treatment neo-squamous tissues from the same level to provide a surrogatemolecular estimate of treatment effect. Methods: Paired pre-treatment BE tissues and post-treatment neosquamous biopsies were obtained from 38 patients treated by RFA (20 patients (3 IMC (prior EMR), 5 HGD, 12 LGD) or EMR (18 patients, 6 IMC, 12 HGD). mRNA expression of 16 genes significantly associated with different BE stages was measured using a multiplex tandem RT-PCR method with preand post-treatment tissues assessed simultaneously. Distal esophageal tissues from 12 patients with no history of GERD or BE were used as normal controls. Results: Endoscopic therapy resulted in a highly significant change in median values towards the normal squamous mucosa expression profile for all 16 genes on unpaired analysis. This change remained significant for 12 genes by paired analysis (t test). The only significant treatment differences in pre-post mean difference in gene expression were greater changes for RFA compared to EMR for 5 genes. The neosquamous expression profile was significantly different to the normal control profile for 9 of 16 genes. Conclusions: Both RFA and EMR result in marked changes in mRNA expression, with replacement of the highly disordered Barrett's dysplasia or IMC expression profile with a more "normal" expression profile. The neosquamous mucosa was significantly different to the normal control squamous mucosa for most genes but the significance of this finding is uncertain and may reflect confounding factors.