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Background Nestin, a class VI intermediate filament protein of the cytoskeleton, and CD34, a transmembrane phosphoglycoprotein, are markers of progenitor cells. This study aimed to evaluate their expression and clinical significance in colorectal cancer. Methods A clinically annotated tissue microarray, including 599 patients with colorectal cancer, was analyzed by immunohistochemistry. Furthermore, nestin and CD34 correlations with HIF-1a and a panel of cytokines and chemokines were assessed using quantitative reverse transcription PCR and The Cancer Genome Atlas dataset. Results Expression of nestin and CD34 was observed only in the tumor stroma. Patients displaying high expression of nestin and CD34 demonstrated higher rates of T1 and T2 tumors (p = .020), lower vascular invasion (p < .001) and improved 5-year overall survival (65%; 95% CI = 55-73 vs 45%; 95% CI = 37-53) after adjusting for clinicopathological characteristics (HR: 0.67; 95% CI = 0.46-0.96). A moderate to strong correlation (r = 0.37-0.78, p < .03) of nestin and CD34 was demonstrated for the following markers; HIF-1 alpha, CD4, CD8, FOXP3, IRF1, GATA3, CCL2, CCL3, CXCL12 and CCL21. Conclusions Combined expression of nestin and CD34 expression is associated with better overall survival possibly by modulating a favorable immune response.

Supplemental Material, Supplemental_tables for Tumor Infiltration by OX40+ Cells Enhances the Prognostic Significance of CD16+ Cell Infiltration in Colorectal Cancer by Fabian Haak, Isabelle Obrecht, Nadia Tosti, Benjamin Weixler, Robert Mechera, Silvio Däster, Marco von Strauss, Tarik Delko, Giulio C. Spagnoli, Luigi Terracciano, Giuseppe Sconocchia, Markus von Flüe, Marko Kraljević and Raoul A. Droeser in Cancer Control

Prostate cancer represents the second leading cause of cancer‐related death in men. T‐cadherin (CDH13) is an atypical GPI‐anchored member of the cadherin family of adhesion molecules. Its gene was reported to be downregulated in a small series of prostate tumours. T‐cadherin protein expression/localisation in prostate tissue has never been investigated. The purpose of our study was to analyse CDH13 gene and protein levels in large sets of healthy and cancer prostate tissue specimens and evaluate CDH13 effects on the sensitivity of prostate cancer cells to chemotherapy. Analysis of CDH13 gene expression in the TCGA RNAseq dataset for prostate adenocarcinoma (N = 550) and in tissue samples (N = 101) by qPCR revealed weak positive correlation with the Gleason score in cancer and no difference between benign and malignant specimens. Immunohistochemical analysis of tissue sections (N = 12) and microarrays (N = 128 specimens) demonstrated the presence of CDH13 on the apical surface and at intercellular contacts of cytokeratin 8‐positive luminal cells and cells double‐positive for cytokeratin 8 and basal marker p63. T‐cadherin protein expression was markedly upregulated in cancer as compared to benign prostate hyperplasia, the increase being more prominent in organ‐confined than in advanced hormone‐resistant tumours, and correlated negatively with the Gleason pattern. T‐cadherin protein level correlated strongly with cytokeratin 8 and with an abnormal diffuse/membrane localisation pattern of p63. Ectopic expression of CDH13 in metastatic prostate cancer cell line DU145 reduced cell growth in the presence of doxorubicin. We conclude that CDH13 protein, but not its gene expression, is strongly upregulated in early prostate cancer, correlates with changes in luminal/basal differentiation and p63 localisation, and promotes sensitivity of cancer cells to doxorubicin. These data identify CDH13 as a novel molecule relevant for prostate cancer progression and response to therapy.

Correlation between MAGE-A10 expression and platinum sensitivity in ovarian cancer patients

Correlation between MAGE-A1 and MAGE-A4 expression and overall survival in patients with high grade endometrial cancer

Aims. A trend towards local excision of early rectal cancers has prompted us to investigate if immunoprofiling might help in predicting lymph node involvement in this subgroup.Methods. A tissue microarray of 126 biopsies of early rectal cancer (T1 and T2) was stained for several immunomarkers of the innate and the adaptive immune response. Patients’ survival and nodal status were analyzed and correlated with infiltration of the different immune cells.Results. Of all tested markers, only CD8 (P=0.005) and TIA-1 (P=0.05) were significantly more frequently detectable in early rectal cancer biopsies of node negative as compared to node positive patients. Although these two immunomarkers did not display prognostic effect “per se,” CD8+ and, marginally, TIA-1 T cell infiltration could predict nodal involvement in univariate logistic regression analysis (OR 0.994; 95% CI 0.992–0.996;P=0.009and OR 0.988; 95% CI 0.984–0.994;P=0.05, resp.). An algorithm significantly predicting the nodal status in early rectal cancer based on CD8 together with vascular invasion and tumor border configuration could be calculated (P<0.00001).Conclusion. Our data indicate that in early rectal cancers absence of CD8+ T-cell infiltration helps in predicting patients’ nodal involvement.

The aim of this study was to assess the expression of cancer testis (C/T) antigens MAGE-A1 and MAGE-A4 in high grade endometrial cancer, as well as to investigate their possible prognostic relevance in patients with endometrial cancer

Recent studies indicate that ER/PR/HER-2-negative (triple-negative, TN) breast cancers may be "CTA-rich" tumors, suggesting the possibility of CTA-based cancer vaccines as a treatment option for patients bearing these tumors. MAGE-A10 together with NY-ESO-1 is probably the most immunogenic CTA, representing a potentially highly attractive target of active specific immunotherapies. Paraffin-embedded tumor sections were collected retrospectively from 165 breast cancer patients diagnosed between 2002 and 2003. Immunohistochemical staining for MAGE-A10 and NY-ESO-1 was performed. The expression of MAGE-A10 and NY-ESO-1 was correlated with other clinicopathological variables. MAGE-A10 expression (score ≥ 2+) was detected in 105/164 (64%), and NY-ESO-1 expression (score ≥ 2+) was observed in 14/164 (8.5%) patients. No correlation between MAGE-A10 and NY-ESO-1 expression and tumor size, tumor grade, Ki-67 and lymph nodes status was detectable. MAGE-A10 expression was significantly associated with ER-negative (P = 0.002), PR-negative (P = 0.002) and HER-2-negative (P = 0.044) tumors. We clearly showed that MAGE-A10 is frequently expressed in the group of TN patients, where the majority (85.7%) of tumors express this CTA. Because of limited therapeutic options for the triple-negative breast cancer, the frequent expression of MAGE-A10 CTA in these cancers may offer the opportunity for a much needed additional treatment for this group of patients.

To evaluate MAGE tumor-associated antigen (TAA) expression in an extensive panel of normal and neoplastic tissues.TAAs of the MAGE family represent targets of active specific immunotherapy. Limited-size studies indicate that they are expressed in normal testis and tumors of different histologies. High-throughput tissue microarray (TMA) technology and MAGE TAA-specific monoclonal antibodies now allow us to comprehensively evaluate their expression in large numbers of tissues and to address clinical correlations.A TMA containing 3,520 samples from 197 different tissues and a non-small-cell lung cancer TMA including 301 specimens were stained using the MAGE TAA-specific monoclonal antibody 57B. For patients with squamous cell carcinoma of the lung, the dichotomous result (positive vs. negative) of MAGE TAA staining was used as a predictor variable along with other covariates in proportional hazard regression analysis of tumor-specific survival.MAGE TAAs are expressed with frequencies ranging between 22.7% (larynx) and 50% of cases (lung) in squamous cell carcinomas from different anatomic areas and in large cell carcinomas of the lung (37.9%). The authors provide here the first description of MAGE TAA expression in basalioma (48.1%). To investigate the clinical significance of MAGE expression in a frequently positive tumor type, a non-small-cell lung cancer, TMA was then studied. In this TMA 43.2% of tumors were 57B positive. In patients with squamous cell carcinoma, MAGE TAA positivity was significantly correlated with a shorter tumor-specific survival in the proportional hazard regression analysis model.These data suggest novel potential therapeutic indications in different types of cancers. In lung squamous cell carcinoma, the significant association of MAGE TAA expression with poor prognosis suggests that patients with 57B-positive tumors may benefit from early, specific immunotherapy procedures.

Aim. The aim of this study is to assess the expression of cancer testis (C/T) antigens in the TNBC group, expression of topoisomerase 2-alpha (TOPO2A), basal-like (BL) immunophenotype defined by basal markers (CK5/6, CK14, and EGFR), BL morphology and conventional clinicopathologic factors as well as to demonstrate their prognostic relevance with respect to this group of tumours. Methods. The study includes 83 patients who underwent surgery between January 2003 and December 2009. Slides were stained immunohistochemically with CK5/6, CK14, EGFR, Ki-67, TOPO2A, MAGE-A1, MAGE-A10, NY-ESO and multi-MAGE. Survival-time and multivariate survival analyses were performed for the purpose of identifying prognostic markers for tumours with more aggressive behaviour. Results. Of the 83 triple negative tumours, 55 (66.3%) have BL immunophenotype and 40 (48.2%) BL morphology. MAGE-A1 show the most frequent expression (69.0%), followed by multi- MAGE (58.0%), NY-ESO (27.1%) and MAGE-A10 (13, 2%). MAGE-A10 expression is significantly correlated with tumour size (p=0.026). The expressions of MAGE-A1, MAGE-A10 and multi- MAGE are significantly correlated with clinical stage (p=0.024, p=0.041, p=0.031, respectively). A significant correlation has been found between the expressions of MAGE-A10 and NY-ESO and the expression of TOPO2A (p=0.005, p=0.013). There is no significant correlation between the expression of C/T antigens and disease-free survival (DFS) or overall survival (OS). Conclusions. High expression levels of MAGE and NY-ESO observed in TNBC are of potential clinical relevance, especially in the adjuvant setting of treatment, and may be of therapeutic value as a vaccine-based treatment in the TN group of breast tumours for which therapeutic options are limited.

We have explored MICA/B expression and its relationship with innate inflammatory infiltrate in renal cell carcinoma (RCC). The expression of MICA/B, CD16, CD56, and CD68 in 140 RCC lesions contained in a tissue microarray (TMA) was investigated by immunohistochemistry. MICA/B gene and protein expressions in Caki-1 cells were analyzed by reverse transcription-polymerase chain reaction and flow cytometry, respectively. Natural killer (NK) cells were studied by flow cytometry. All the RCC lesions (n = 140) were MICA/B-positive. MICA/B was mainly expressed in the cytoplasm of tumor cells, whereas stromal cells were negative. Renal cell carcinoma lesions showed low NK cell infiltration, although they were rich in CD16(+)CD56(-) cells, strongly resembling macrophages. CD16(+) macrophage infiltration was more frequently detectable in metastatic lesions compared with primary tumors (P = .0223) and was associated with poor RCC differentiation (P = .007). To investigate mechanisms potentially underlying the lack of NK cells infiltration into MICA/B-positive RCC lesions, we used Caki-1 RCC cells. Caki-1 expressed MICA and MICB genes. However, MICA protein was not detectable in Caki-1 cells, whereas MICB protein was detectable in their cytoplasm and on the cell membrane. Coculture of peripheral blood mononuclear cells with Caki-1, K562, HCT116, respectively, resulted in CD56(+)CD16(+) NK cells deletion without affecting CD56(+)/CD16(-) NK subset and immature NK cells generated in vitro from CD34(+) cells. Natural killer cell apoptosis seemed to be preferentially triggered by cancer cells because HLA-A0201(+) NK cells were only marginally affected by allogeneic HLA-A0201(-) peripheral blood mononuclear cells. Caki-1 cell-mediated NK cell apoptosis was reduced by an anti-beta(2)-integrin (CD18) monoclonal antibody but was NKG2D-, granule exocytosis-, and caspase-independent.

Expression of cancer/testis antigens was analyzed by immunohistochemistry in medullary breast cancer and correlated with the standard clinical and pathohistological parameters.

Besides their variable presence in fetal and adult germ cells, CT antigens have occasionally been detected in placental tissue. However, these data are scarce and solely based on mRNA analyses; nothing is known about their presence at the protein level. Here, we analyzed the expression of various CT antigens in placental tissues from gestational age week 5 to week 42 using monoclonal antibodies to various antigens of the MAGE-A and -C families, NY-ESO-1, as well as GAGE. We show that CT antigen expression in placenta varies widely for the various antigens, ranging from completely negative to abundant. Since little is known about the function and biology of CT antigens, interpretation of this highly variable expression pattern is purely speculative. However, our data indicate that the various CT antigens have different functions during placental development.

Skupina gena MAGE pokazuje izraženost u različitim tumorima a od normalnog tkiva nalaze se samo u testisima i placenti.Danas se pokušavaju upotrijebiti kao ciljevi liječenja u imunoterapiji.Istraživanje je pokazalo da su imunohistokemijski jače izraženi u uznapredovalim karcinomima debelog crijeva što znači da su ti bolesnici pogodni za primijenu specifične imunoterapije.

Aim To evaluate the possible prognostic role of the expression of MAGEA4 and NY-ESO-1 cancer/testis antigens in women diagnosed with invasive ductal breast cancer and determine the expression of HER-2 antigen. Methods The expression of MAGE-A4, NY-ESO-1, and HER-2 antigens was evaluated immunohistochemically on archival paraffi n-embedded samples of breast cancer tissue from 81 patients. All patients had T1 to T3, N0 to N1, M0 tumors and underwent postoperative radiotherapy and, if indicated, systemic therapy (chemotherapy and hormonal therapy). Th e antigen expression in women who were disease-free for 5 years of follow up (n = 23) was compared with that in women with either locoregional relapse (n = 30) or bone metastases (n = 28). Patient survival aft er 10 years of follow up was assessed. Results The three groups of women were comparable in terms of age, type of operation, tumor size, tumor grade, number of metastatically involved axillary lymph nodes, Nottingham prognostic index (NPI), progesterone receptor (PR) status, and adjuvant hormonal therapy. Estrogen receptors (ER) were positive in 13 women in the 5-year relapse-free group vs 8 in locoregional relapse and 7 in bone metastases group (P = 0.032). Th ere were significantly fewer women who received adjuvant chemotherapy in the 5-year relapse-free group than in other two groups (7 vs 23 with locoregional relapse and 25 with bone metastases ; P

Objective: To investigated immunohistochemical expression of MAGE-A and NY-ESO-1/LAGE-1, cancer testis antigens (CTA) in penile carcinoma tissue. Methods: 30 penile carcinoma samples from patients undergoing penile amputation at the Urology Clinics at the Zagreb Clinical Hospital Center and Clinical Hospital "Sestre milosrdnice" from 1997 to 2004 were investigated in this study. Three monoclonal antibodies were used for immunohistochemical staining: 77B for MAGE-A1, 57B for multi-MAGE-A and D8.38 for NY-ESO-1 expression. Results: Expression of MAGE-A1 was observed in 13% of carcinoma samples, whereas both multi-MAGE-A and NY-ESO-1 stained 97% of samples (p

MAGE family gene products are tumor associated antigens (TAA) recognized by HLA restricted specific T cells. Agents inducing DNA demethylation, an event typically detectable in cellular de-differentiation processes, were shown to induce the expression of MAGE genes. Specific monoclonal antibodies now allow the detection of MAGE gene products in large series of archival paraffin embedded tumor sections. By using a monoclonal antibody specific for MAGE family gene products we have studied the expression of these TAA in a group of 144 patients bearing invasive ductal breast cancers. Immunohistochemical data were correlated with tumor differentiation, lymphatic vessels invasion, lymphnode metastases, estrogen receptor expression, peritumoral lymphocyte infiltration and intratumoral necrosis. MAGE immunoreactivity was undetectable in healthy tissues. In poorly differentiated cancers positive staining was observed in 30/63 cases (47.6%) as compared with 13/51 (25.4%) and 5/30 (16.6%) in moderately and well differentiated tumors, respectively (p

Cell membrane gangliosides have been shown to be involved in a number of biological processes including cell adhesion, signal transduction and ligand receptor interactions. In this paper we analyzed the effects of a mixture of bovine brain gangliosides, currently in clinical use, on cell mediated immune responses in vitro. We show here that exogenous gangliosides inhibit mitogen and alloantigen induced lymphoproliferation. On the other hand effects on antigen induced blastogenesis were exquisitely dose dependent in that while high doses of gangliosides inhibited lymphoproliferation, probably by interfering in interleukin 2 receptor interactions, lower doses significantly enhanced antigen induced responsiveness. We also report that gangliosides inhibit the generation of lymphokine activated killer cells. Altogether, these data underline the immunoregulatory potential and the polymorphism of effects of exogenous gangliosides. © 1990.

BACKGROUND Colorectal cancer (CRC) infiltration by adaptive immune system cells correlates with favorable prognosis. The role of the innate immune system is still debated. Here we addressed the prognostic impact of CRC infiltration by neutrophil granulocytes (NG). METHODS A TMA including healthy mucosa and clinically annotated CRC specimens (n = 1491) was stained with MPO and CD15 specific antibodies. MPO+ and CD15+ positive immune cells were counted by three independent observers. Phenotypic profiles of CRC infiltrating MPO+ and CD15+ cells were validated by flow cytometry on cell suspensions derived from enzymatically digested surgical specimens. Survival analysis was performed by splitting randomized data in training and validation subsets. RESULTS MPO+ and CD15+ cell infiltration were significantly correlated (p

T cadherin (T cad) is gaining recognition as a determinant for the development of incipient invasive squamous cell carcinoma (SCC). However effects of T cad expression on the metastatic potential of SCC have not been studied. Here using a murine model of experimental metastasis following tail vein injection of A431 SCC cells we report that loss of T cad increases both the incidence and rate of appearance of lung metastases and reduces survival. T cad silenced SCC metastases are highly disordered with evidence of single cell dissemination away from main foci whereas SCC metastases overexpressing T cad develop as compact tightly organized sheets. SCC adhesion to vascular endothelial cells (EC) in culture and transmigration across cultures of vascular and lymphatic EC is increased for T cad silenced SCC and decreased for T cad overexpressing SCC. Confocal microscopy shows that T cad silenced SCC adherent on EC display an elongated morphology with long thin extensions and a high degree of intercalation within the EC monolayer whereas SCC overexpressing T cad formed poorly spread multicellular aggregates that rather remain on the outer surface of the EC monolayer. Our data suggest that loss of T cad can increase metastatic potential and aggressiveness of SCC possibly due to facilitating arrest and extravasation through the vascular wall and/or more efficient establishment of metastases in the new microenvironment.

// Silvio Daster 1, * , Nunzia Amatruda 1, 2, * , Diego Calabrese 2 , Robert Ivanek 2 , Eleonora Turrini 3 , Raoul A. Droeser 1 , Paul Zajac 1, 2 , Carmela Fimognari 3 , Giulio C. Spagnoli 1, 2 , Giandomenica Iezzi 1, 2 , Valentina Mele 1, 2, ** , Manuele G. Muraro 1, 2, ** 1 Department of Surgery, University Hospital Basel, Basel, Switzerland 2 Department of Biomedicine, University of Basel, Basel, Switzerland 3 Department for Life Quality Studies, University of Bologna, Rimini, Italy * These authors have contributed equally to this work ** Shared senior authorship Correspondence to: Valentina Mele, email: valentina.mele@usb.ch Manuele G. Muraro, email: manuele.muraro@usb.ch Keywords: multicellular tumor spheroids, three-dimensional culture, tumor model, hypoxia, necrosis Received: April 19, 2016 Accepted: September 19, 2016 Published: December 10, 2016 ABSTRACT Culture of cancerous cells in standard monolayer conditions poorly mirrors growth in three-dimensional architectures typically observed in a wide majority of cancers of different histological origin. Multicellular tumor spheroid (MCTS) culture models were developed to mimic these features. However, in vivo tumor growth is also characterized by the presence of ischemic and necrotic areas generated by oxygenation gradients and differential access to nutrients. Hypoxia and necrosis play key roles in tumor progression and resistance to treatment. To provide in vitro models recapitulating these events in highly controlled and standardized conditions, we have generated colorectal cancer (CRC) cell spheroids of different sizes and analyzed their gene expression profiles and sensitivity to treatment with 5FU, currently used in therapeutic protocols. Here we identify three MCTS stages, corresponding to defined spheroid sizes, characterized by normoxia, hypoxia, and hypoxia plus necrosis, respectively. Importantly, we show that MCTS including both hypoxic and necrotic areas most closely mimic gene expression profiles of in vivo-developing tumors and display the highest resistance to 5FU. Taken together, our data indicate that MCTS may mimic in vitro generation of ischemic and necrotic areas in highly standardized and controlled conditions, thereby qualifying as relevant models for drug screening purposes.

Colorectal cancer (CRC) represents a major cause of cancer related death in different geographic areas. Tumor infiltration by a variety of immune cell types has consistently been observed to be associated with favorable prognosis (1). In particular, CRC infiltration by CD8+ T cells expressing memory and activation markers has repeatedly been reported to represent a favorable prognostic marker, although underlying molecular mechanisms and the antigen specificity of these cells are largely unclear. In sharp contrast, tumor infiltration by myeloid cells has classically been associated with poor prognosis in a variety of cancers of different histological origin (2). However, recent reports underline that granulocytes might participate to anti-tumor immune responses in defined cancer types (3-5). In previous studies, our group has observed that CRC infiltration by myeloid cells expressing CD16 and myeloperoxidase (MPO) is also associated with good prognosis (6;7). Based on this background, I have addressed the role of granulocyte infiltration in CRC immunobiology. By using a clinically annotated tissue microarray (TMA) including over 650 individual CRC, I have explored the prognostic significance of tumor infiltration by CD66b+ granulocytes. I found that CRC infiltration by CD66b+ granulocytes significantly correlates with favorable prognosis. Most importantly however, dense granulocyte infiltration significantly enhances the prognostic significance of CD8+ T cell infiltration in CRC. Taken together these data suggest that a cross-talk of potentially high clinical relevance takes place in CRC. Indeed, immunofluorescence staining of tissue sections from CRC showing evidence of infiltration by lymphocytes and granulocytes indicates that CD8+ and CD66b+ cells are frequently co-localized. Still unclear is whether neutrophils are able to modulate T-cell responsiveness to antigenic challenges (3;8). On the other hand activated T cells have previously been shown to enhance granulocyte survival and functions by the release of cytokines, including TNF-α, IFN-γ and GM-CSF (8). In additional studies I have extensively characterized phenotypic profiles of CRC infiltrating granulocytes, as compared to granulocytes from peripheral blood or infiltrating healthy mucosa adjacent to CRC. Functional studies performed by using tumor-derived and peripheral blood neutrophils indicate that these cells are able to efficiently co-stimulate CD8+ T cell activation induced by T-cell receptor (TCR) triggering, as witnessed by increased expression of CD69 activation marker, proliferation and IFN-γ release. Furthermore, an expansion of cells expressing a “central memory” phenotypic profile was also observed in cultures stimulated in the presence of autologous neutrophils. Furthermore, importantly, malignant transformation in the intestinal mucosa is associated with early translocation of microorganisms from the gut lumen (9). A variety of bacterial strains including Bacteroides fragilis and Fusobacterium nucleatum have been found to be highly represented in CRC (10). Capitalizing on these data, I have investigated the potential significance of the interaction with bacterial strains associated with CRC in the elicitation of the anti-tumor effects of neutrophils. I found that interaction with Fusobacterium nucleatum rapidly induces apoptosis in neutrophils and abrogates their co-stimulatory capacity. Taken together, my results contribute to the identification of CRC microenvironment as typically characterized by a “ménage à trois”, including cancerous cells, the immune system and gut colonizing microorganisms. The nature of the reciprocal interaction of these actors and its outcome are likely to decisively impact on CRC development and progression

Natural killer (NK) cells represent the largest proportion of innate effector lymphocytes and account for 10-15% of total peripheral lymphocytes. In contrast to B- and T- cells, NK cell activation does not depend on clonally rearranged and antigen specific surface receptors. Instead, NK cells rely on an array of activating and inhibitory germ-line encoded receptors. When encountering a target cell, activating and inhibitory signals are integrated and a response is formed immediately. Killer cell immunoglobulin-like receptors (KIR) are thereby critical for determination of NK cell activation. KIR receptors can be either activating or inhibitory in nature. Inhibitory KIR receptors not only regulate NK cell function, but are also involved in a developmental process called “licensing”. During licensing, NK cells which express an inhibitory KIR capable of recognizing HLA develop increased functional competence. Both, inhibition of mature NK cells and NK cell licensing are based on the interaction of inhibitory KIR with their cognate HLA class I ligands encompassing HLA-C and subsets of HLA-B and HLA-A. NK cell “licensing” is still poorly understood and many questions remain open. So far it remains elusive if the quantity of HLA expression is a variable in NK cell licensing. We took advantage of two recently identified polymorphisms affecting the HLA-C expression on the cellular surface rs9264942 and rs67384697. We genotyped 66 healthy blood donors for these polymorphisms and assessed the quantity of HLA-C expression by FACS using an HLA-C specific antibody. In addition, we assessed the presence of the inhibitory KIR receptors KIR2DL1and KIR2DL3 and their corresponding HLA ligands, being two mutually exclusive groups of HLA-C: HLA-C1 and HLA-C2. In subsequent functional analyses, we observed, in agreement with the concept of licensing, increased functionality of KIR2DL1+ and KIR2DL3+ NK cells in donors expressing the corresponding HLA ligand in a dose dependent manner,. The quantity of HLA-C surface expression, however, did not affect the quality of NK cell licensing. A unique opportunity to study NK cell licensing is provided during the first months after hematopoietic stem cell transplantation when the NK cell repertoire is rebuilt. Previous studies found that leukemia patients receiving hematopoietic stem cell transplantation (HSCT) from a haploidentical donor, benefit from a survival advantage if KIR ligands are mismatched and NK cell tolerance is irreversibly broken. Recent studies reported controversial findings concerning a survival advantage and NK cell mediated graft versus leukemia effect (GVL) as a consequence of disturbed NK cell licensing early after HSCT, when NK cells are unselectively equipped with functional capacity. Differences in allogeneic HSCT protocols were discussed as possible reason for the opposing results. We investigated reconstitution of NK cell function and NK cell licensing in 56 patients receiving allogeneic (33) or autologous (23) HSCT during the first six months after transplantation. We found that NK cell licensing was maintained after both kinds of transplantation. However licensing effects were less distinct after allogenic compared to autologous HSCT. Additionally, we identified GvHD and pre-transplant ATG administration as variables associated with less prominent licensing in recipients of allogeneic grafts. Whereas research on inhibitory KIR and NK cell licensing has already influenced treatment modalities of HSCT, much less is known about activating KIR receptors. Ligands to many activating KIR remain unidentified and little is known in which diseases activating KIR may play a role. Recent studies in our lab and elsewhere described associations between KIR genotypes and relative protection from cytomegalovirus (CMV) replication after solid organ transplantation (SOT) and HSCT. We prospectively followed a cohort of 649 patients after SOT, assessed the KIR genotype and recorded common opportunistic viral infections. Subsequent analyses of our data revealed an association of KIR B haplotypes, which encompass many activating KIR, and relative protection from varicella zoster virus (VZV) and a tendency for relative protection from Epstein-Barr virus (EBV) replication. In subsequent analyses we found that centromeric rather than telomeric activating KIR protect from VZV. In contrast, we detected no association between activating KIR genotype and BK polyomavirus (BKPyV) or Herpes simplex (HSV) replication. Besides intrinsic factors such as licensing or expression of activating KIR, extrinsic factors determine antitumor and antiviral activity of NK cells. Early after HSCT when NK cell driven GVL is assumed most effective, prophylactic immunosuppression is given and cellular products are administered including erythrocytes (ERY) and platelets (PLT). During storage, blood products release ectosomes. PLT ectosomes can reduce monocyte and dendritic cell function. Furthermore, they favour differentiation of naïve T-cells into T-regulatory cells. However, little is known about their interaction with NK cells. We assessed phenotypical and functional changes on NK cells after co-incubation with PLT ectosomes in vitro, and found a reduction of NK cell function and activating surface receptors, mediated through TGF-β on PLT ectosomes.

Colorectal cancers (CRCs) show extensive genetic and epigenetic aberrations, including DNA sequence changes, chromosomal alterations and abnormal epigenetic chromatin modifications. These alterations often reflect defects in molecular mechanisms establishing and maintaining (epi-) genome integrity. Epigenetic alterations in CRC are well documented, both, at the levels of histone modifications and DNA methylation. Epigenetic mechanisms play critical roles in the regulation of DNA-templated processes such as transcription, DNA repair but also replication. Shaping higher order chromatin structure, they regulate DNA accessibility and, thereby, assure proper genome function. Although many epigenetic alterations in CRC and other cancers are described and, in specific cases, their role in carcinogenesis established, the origin of and the molecular mechanisms underlying epigenetic instability in CRC remained elusive. The aims of my thesis were to elucidate the molecular basis of DNA methylation aberrations in CRC in a first part, and to investigate mechanisms by which CRC-associated DNA methylation alterations contribute to colorectal carcinogenesis in a second part. A subset of human CRCs is characterized by genome-wide hypermethylation of promoter CpG islands (CGIs) and therefore referred to as displaying a CGI-methylator phenotype (CIMP). Mutational inactivation and metabolic inhibition of the Ten Eleven Translocation (TET) DNA dioxgenases, key factors of active DNA demethylation, have been reported to be causal for DNA hypermethylation phenotypes in leukemias and gliomas. We investigated whether deregulated, TET-mediated DNA demethylation can account for CIMP in CRC. We discovered that TET1 is significantly downregulated, both at the mRNA and protein level, in CIMP-displaying CRCs. Knock-down of TET1 in the non-CIMP CRC cell line SW620 confirmed that the loss of TET1 results in genome-wide CGI hyper- and hypomethylation in cell culture, which, upon xenograft tumor formation, converted into a hypermethylation phenotype. Notably, the loss of TET1 in the CRC cell line resulted in increased promoter methylation and downregulation of MLH1 and CDH1, a characteristic feature of CRC-CIMP. These results thus showed that depletion of TET1 and selection during tumor growth can recapitulate features of CIMP in non-CIMP cancer cells. Thus, TET1 activity in colon epithelial cells contributes to maintenance of cell type specific CGI-methylation patterns and prevents the development of CRC through the CIMP-pathway. Beside cancer-specific defects in the maintenance of CpG methylation, aging constitutes a major source of methylation alterations associated with CRC. Lifestyle-related factors including overweight, aspirin use, hormone replacement therapy (HRT) and smoking have been reported to influence the risk of CRC over long periods of time. We investigated whether these factors exert their effects on cancer risk via modulation of age-related DNA methylation changes. Genome-wide and detailed target-wise methylation analysis in a cohort of 546 healthy women revealed that promoter-associated DNA methylation increases over age. We observed that smoking and obesity promote, whereas aspirin and HRT reduce the genome-wide methylation drift. A significant fraction of promoter-associated CpG sites, whose age-related hypermethylation was modified by lifestyle factors, also occurred hypermethylated in CRC. Lifestyle factors affected the methylation drift of CRC-related CpGs concordant with the effects of these factors on CRC risk. Our results suggest that the effect of the investigated lifestyle factors on CRC-risk can be accounted for by the modulation of age-related DNA methylation changes. The promoter methylation of cancer-relevant genes detectable in the healthy colorectal mucosa allows the definition and monitoring of early epigenetic events in colorectal carcinogenesis. To explore this possibility, we selected five genes (FOXF1, CA4, NPY1R, GREM1 and IFITM1) downregulated in colorectal adenomas and quantified their promoter methylation levels in CRC tissue as well as in the matched healthy appearing colonic mucosa. We included MLH1 and MGMT, two genes with established roles in colorectal carcinogenesis, in this analysis. With different incidence all seven markers were found hypermethylated in 106 CRC samples tested. Interestingly, methylation of the two novel markers CA4 and GREM1 associated with CRC-CIMP. We found that these two markers are methylated at low levels also in cancer-associated mucosa as well as in colonic mucosa from healthy individuals, as it was reported previously for the CIMP marker MLH1. These results thus suggest that features of CIMP-specific DNA methylation are present and, hence, detectable already in the mucosa of healthy individuals. DNA hypermethylation in gene promoters often interferes with transcription of the associated gene. We identified SHOCA-2 as a regulator of Epidermal Growth Factor Receptor (EGFR) signaling and observed its loss of expression in advanced CRCs. The loss of SHOC-2 expression was associated with different genetic and epigenetic alterations. We detected chromosomal deletions affecting the SH2D4A gene (encoding for SHOCA-2) as well as mutations in the coding region of the gene. Finally, we found the cancer-specific hypermethylation of two CpG sites in the 5´-untranslated region of SH2D4A, one of which overlapped with a binding site for the transcription factor Sp1. We found that methylation of this CpG site in the SH2D4A sequence context interferes with Sp1 binding in vitro and therefore might be responsible for SHOCA-2 expression loss in some CRCs. This describes a molecular mechanism by which site-specific DNA methylation may contribute to cancerous transformation.

In an increasing number of cancers, CSCs have been defined on the basis of the self-renewal and tumor initiation capacity by functional assays. It has been also suggested that CSC populations might be responsible for chemo- and radio-therapy resistance within tumors and ultimately for the post-therapeutic tumor recurrence. The identification of markers identifying CSC is fundamental for the validation of the CSC paradigm and for the development of new CSC-specific drugs and novel therapeutic approach. CD133, CD44 and CD166 have been proposed as putative CSC markers in CRC. These findings have opened the field for an extensive validation of the markers and their use for the development of specific anti-CSC therapy. In this work, I addressed a) the prognostic relevance of the expression of CSC surface markers in CRC clinical specimens, b) the “in vivo” tumorigenicity of primary CRC derived cells, as related to their expression of putative CSC surface markers, c) the possibility of using cells derived from established CRC cell lines expressing CSC surface markers as CSC cellular model, and finally d) the development of innovative culture models of potential relevance for the screening of anti CRC compounds. I have analyzed the surface markers expression in correlation with stem cell-like functional features, but no consistent results was found confirming stemness property associated with expression of those markers. These results obviously question the validity of putative surface CRC-SC markers. Taken together these data might suggest that their expression and CSC functional features might be associated with some degree of plasticity, potentially related to tumor microenvironmental characteristics being lost in conventionally cultured tumor cell lines and in primary tumor derived cell suspensions. Based on this background I have investigated the possibility to perform 3D culture of CRC cell lines to assess whether these systems might provide useful insights for the interpretation of our data. My findings clearly document the plasticity of gene expression profiles of cultured CRC cells depending on their three-dimensional architectures. Most importantly, I demonstrate that major gene expression modulation events only occur when culture in 3D spheroids is associated with ischemia and necrosis. I also showed that the gene expression of putative CSC markers increases with the occurrence of ischemia and necrosis in the core of a 3D spheroid.

The thymus is the primary lymphoid organ responsible for T-cell development and selection, which requires a specialised stromal microenvironment. The thymic stroma consists mainly of thymic epithelial cells (TEC), which can be divided into cortical (c) and medullary (m) TECs, mediating each different functions during T-cell development. The organisation of the thymus compartments is crucial for T-cell development. Therefore it is important to understand the mechanisms behind thymus development as defects in thymus function can lead to immunodeficiency or autoimmunity. In the context of thymus organogenesis, Foxn1 serves as a master transcription factor essential for TEC development. The loss of Foxn1 constitutes then a molecular cause for athymia as the molecules absence blocks TEC growth and differentiation. Consequently the epithelial cells adapt another cell fate and the thymus rudiment fails to attract lymphoid precursors. Mice with either a reduced or time restricted Foxn1 expression at the onset of thymus organogenesis displayed a severely disturbed thymus development. Though able to develop into cTEC and mTEC, they remain in an immature state as demonstrated by the absence of mature TEC. Similar as in congenital athymic nude mice, devoid of Foxn1 expression, also common bipotent progenitor TEC were detected. T-cell development was impaired resulting consequently in severe peripheral lymphopenia, which only slightly mitigated with progressive age. Importantly the peripheral T-cells in both mouse models differ from that of nude mice, in that cells with effector and regulatory functions were detected. Furthermore inadequate Foxn1 expression lead to colitis and early death. Bowel inflammation was associated with an increase in Th1 polarised CD4+ T-cells among intraepithelial and lamina propria lymphocytes. In aggregate this study showed that TEC differentiation and maintenance depends on both a continuous and adequate Foxn1 expression, as short-term or low level Foxn1 expression were insufficient to sustain regular thymus development and function. Inadequate expression of Foxn1 expression resulted consequently in the loss/absence of a regular thymus microenvironment and selection of an autoreactive T-cell repertoire causing auto-immune colitis.

The tumor microenvironment contributes significantly to the clinical prognosis, especially in CRC. Infiltration by cells from the adaptive immune system (e.g. CD8) has been linked to a better survival independently of TNM-stage. The complex tumor microenvironment is not well mirrored by conventional in vitro tumor assays where response to drug or treatment is evaluated on tumor cell lines monolayers. In has been proposed that 3D tumor models could at least to some extent better represent the in-vivo tumor microenvironment. In this work we aimed therefore not only at the characterization of the CRC- microenvironment in regard to innate and adaptive immunity but also to engineer innovative in vitro models of CRC better reflecting in-vivo tumor response to treatment. Using tumor microarray (TMA) technology we found that an infiltration by cells with neutrophil phenotype expressing high MPO positivity was associated with a favorable prognostic effect and correlated with CD8 infiltration. As tumoricidal activity of CD8-T-cells has been described to be influenced by immunological checkpoints engagement we evaluated in another TMA study the expression of PD-L1 on colorectal cancer specimens. PD-L1 expression on CRC correlated with CD8 infiltration but, interestingly, was associated with a good survival. This intriguing role of the microenvironment composition on the survival of colorectal cancer patients lead us then to start working on an innovative perfused 3D tumor model to better mimic the complexity of cancer tissue in-vivo. By using cancer cell lines we could establish a model, which had phenotypical but as well functional properties similar to xenografted tumors. Most importantly the response to standard treatments, were also similar. As this perfused tumor model only partially mimics the complex tumor microenvironment, an ultimate goal remains the culture of primary colorectal cancer specimens. By using the perfused u-tube-bioreactor and adaption of protocol using collagen-sandwich we could with a high efficiency keep tumor tissue alive and proliferating. In our studies on CRC microenvironment we could observe that activated neutrophils, somehow unknown and unappreciated players in tumor immunology, contributed significantly to a better survival. On the other hand the immune-regulatory checkpoint ligand PD-L1 has been associated with improved survival in CRC in contrast to melanoma or breast-cancer. This could be regarded as marker of an ongoing-immune response because we observed a correlation between CD8 cells and IFN-γ gene expression. Engineering the complex tumor microenvironment by a perfused 3D bioreactor able to generate large tumor-tissues in short time could be helpful as tool for further phenotypical and functional screenings. Additionally, we could observe a significant impact of perfusion on survival and proliferation of tumor cells within primary tumor fragments.

The notion that tumour initiation and heterogeneity might be driven by small population of tumour-initiating cells (TIC) has gained high significance since the pioneering identification of TIC in leukaemia. This has led to a worldwide research effort to further identify TIC in solid tumours. In prostate cancer (PCa), however, demonstration of the existence and identification of TIC have been hampered by a lack of consistent in vitro and in vivo models. Chapter I of this thesis presents several models of human PCa and their respective significance to study TIC in PCa. This chapter also describes my attempts to establish more relevant models by generating and characterising short-term primary cultures derived from clinical PCa specimens. TIC are thought to share some properties of normal stem cells and to express genes typically expressed by embryonic stem cells. Based on this hypothesis, I investigated the expression of stemness-associated genes in PCa. Findings are presented in Chapter II of the thesis and described in the published manuscript entitled “Klf4 transcription factor is expressed in the cytoplasm of prostate cancer cells”. Additionally, same as normal stem cells, TIC might display high activity of aldehyde dehydrogenase (ALDH) enzyme. Chapter III reports the characterisation of a cell subset exhibiting high ALDH activity in PCa. In particular, features, prevalence and clinical significance of these cells in PCa are presented in the manuscript entitled “Characterization and clinical relevance of ALDHbright populations in prostate cancer”. Taken together, my thesis highlights the complexity of the TIC concept and the urgent need for more accurate models, paving the way for further studies aiming at identifying TIC in human PCa.

Breast cancer is the most common malignancy among females and the fifth most common cause of cancer death worldwide. The research for biomarker discovery for the diagnosis and prognosis of patients with breast cancer has been going on for decades, yielding only a few major breakthroughs. Our increasing understanding of cancer biology, including genetic, molecular and cellular mechanisms, and epigenetic background is now providing objectives for the early detection of some malignancies including breast cancer. Such a progress has a direct impact on current activities dedicated to the search for sensitive and specific biomarkers for the early detection and diagnosis of cancers. In the present study, to find an efficient method for analyzing the methylation profile in breast cancer, we developed a method that allows for the simultaneous detection of multiple targets CpG residue by employing thymidine-specific cleavage on MALDI-TOF MS. Using this method we determined quantitative methylation changes of 22 candidate genes in breast cancer tissues. Firstly, we analyzed the methylation status of a total of 42,528 CpG dinucleotides on 22 genes in 96 different paraffin-embedded tissues (48 breast cancerous tissues and 48 paired normal tissues). In this study, 10 hypermethylated genes (APC, BIN1, BMP6, BRCA1, CST6, ESRb, GSTP1, P16, P21 and TIMP3) were identified to distinguish between cancerous and normal tissues according to the extent of methylation. These hypermethylated genes may serve as biomarkers for clinical molecular diagnosis and targeted treatments of patients with breast cancer. To achieve a gene panel for developing a breast cancer blood-based test according to the pathologic methylation changes, we quantitatively assessed the DNA methylation proportion of 31,248 CpG sites on 10 candidate genes (APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P16, P21 and TIMP3). The number of 126 samples consisting of two different cohorts was used (first cohort: plasma samples from breast cancer patients and normal controls; second cohort: triple matched samples including cancerous tissue, matched normal tissue and serum samples). Circulating cell free methylated DNA of the 8 tumor suppressor genes (TSGs) was significantly higher in patients with breast cancer compared to normal controls. Using eight genes as a panel to develop a blood-based test for breast cancer, a sensitivity and specificity of more than 90% could be achieved in distinguishing between tumor and normal samples. Presented data is promising to design a gene panel and develop a blood-based screening method for breast cancer which relies on pathologic methylation changes. Analyzing the relative telomere length in paired breast cancer and matched normal tissue demonstrated a significant shortening of telomere regions in the tumor compared to paired adjacent normal tissues. Moreover, promoter hypermethylation of the p16/Rb and p53/p21 pathways showed significant correlation with telomere shortening. The results suggested that inactivation of p16/Rb and/or p53/p21 pathways by hypermethylation may be linked to critical telomere shortening, leading to genome instability and ultimately to malignant transformation. Thus, shortened telomere length and hypermethylation of p53, p21 and p16 promoters might serve as biomarkers in breast cancer. Epigenetic changes can be pharmacologically reversible and could be a useful target to develop new therapeutic strategies for cancer therapy. Reversal hypermethylation of silenced genes/miRNAs inhibiting malignant phenotypes is increasingly being targeted for cancer therapy and prevention strategies. In our study, the therapeutic value of detected hypermethylated tumor suppressor genes in different subtypes of breast cancer was assessed after treatment with a demethylating agent (5-aza-2-deoxycytidine; DAC). Additionally, we investigated multidimensional pan-omics models to predict effects of DAC treatment at the level of the genome, epigenome and proteome-wide alterations in highly aggressive and non-aggressive subtypes of breast cancer. The results provided early and late effects of this treatment in 3-dimentional follow-up omics model as cancer and non-cancer specific changes that may correlate with particular steps in breast neoplasm including cell proliferations, cell/tissue invasion, oncogenesis, angiogenesis, apoptosis, neoplasm metastasis and senescence. Additionally, beside the activation of epigenetically suppressed TSGs, we also showed significant down-regulation of some miRNAs with oncogenic functions in breast cancer cell lines as well as up-regulation of some other miRNAs with tumor suppressor functions that highlights the usefulness potential of a miRNA-based therapy in breast cancer. The obtained results provided a rationale for developing therapeutic strategies based on reactivation of epigenetically silenced genes/miRNAs in breast cancer.

Background Recombinant poxviruses expressing tumor associated antigens (TAAs) are evaluated since 20 years as immunogenic vaccine vector in clinical trials. Some caveats for using recombinant viral vector are due to either prior systemic immunity to poxviruses or immunodominance of viral antigens which may reduce the induction of immune response against weaker tumor antigens. To address this issue, we developed a recombinant vaccinia virus (VV) expressing herpes simplex virus (HSV) type I protein ICP47. This protein down-regulates MHC class-I antigen presentation by blocking the transporter associated with antigen processing (TAP), which translocates peptides, generated by proteasomal protein degradation, into the endoplasmic reticulum for loading onto MHC class I molecule. Methods HSV-US12 gene, coding for ICP47 was introduced into wild type vaccinia virus (VV) and into r.VV expressing MART-1/Melan-A27-35 HLA-A201 ER-targeted epitope. Following infection with non-replicating recombinant virus, effect of ICP47 expression on cell surface MHC-class-I, MHC class-II and co-stimulatory molecules was characterized by antibody staining and FACS analysis. Human T-lymphocytes were stimulated in vitro with autologous CD14+ cells infected with r.VV-US12, r.VV-Mart-US12 or control virus. Responsiveness of specific CD8+ and CD4+ T cells to viral proteins and to recombinant epitopes was monitored by MHC-multimer staining and cytokines (IFNγ & IL-2) expression analysis. In order to highlight in vitro the immunogenic advantage provided by ICP47 blockade of viral epitopes, T cells were pre-sensitized with WT-VV infected APC and then used as responders to r.VV-Mart-US12 or r.VV-Mart infected APC. Results Cells infected with HSV-US12-r.VV, demonstrated a decreased ability of presenting MHC class-I antigens to CD8+ T cells whereas MHC-class-II restricted presentation to CD4+ T cells remained unaffected. Cell surface expression of CD80 as a costimulatory molecule and CD44 as cell adhesion molecule was not affected in cells infecied with VV expressing ICP47. Co-expression of ER-Melan-A/Mart-127-35 appeared to partially compensate for the ICP47 related surface MHC class-I down-regulation and preserve a strong capacity to induce CTL response against the TAA derived peptide. The differential prime-stimulation for Mart epitope, in conditions where clearance of infected APC by VV specific CTL could become a limiting factor, showed significantly enhanced CTL responses to Mart stimulation as characterized by IFN-γ and IL-2 gene expression in cultures primed with r.VV-Mart-US12 as compared to cultures primed with r.VV-Mart. Conclusion Thus, viral vectors expressing ICP47 confirmed a diminished TAP-dependant processing of endogenous class-I restricted epitopes while the immunogenic potential of recombinant epitopes directly targeted to the ER was enhanced. Such reagents could become of high relevance especially in multiple-boost vaccine protocol required in cancer immunotherapy.

s: Glucocorticoids have an effective anti-inflammatory, anti-proliferative and immunomodulatory activity. Therefore this class of drugs is used worldwide for the treatment of inflammatory diseases and to prevent rejection following organ transplantation. Inhaled glucocorticoids are the cornerstone treatment of asthma and advanced chronic obstructive pulmonary disease (COPD). The new long acting glucocorticoids mometasone and Ciclesonide have recently been introduced for the treatment of asthma. The aim of these studies is to assess the kinetics and molecular pathways of glucocorticoid receptor (GR) activation and traffic in human lung fibroblasts and bronchial smooth muscle cells. Furthermore the effects of new long acting glucocorticoids on GR activation, complex formation with the transcription factor C/EBP and its isoforms proliferation will be evaluated. Glucocorticoids enter the cytosol of cells by diffusion and bind to an intracellular specific receptor, the glucocorticoid receptor (GR), inducing a conformational change and leading thereby to its activation. The active GR translocates into the nucleus, where it binds to its cognate DNA recognition sequence, the glucocorticoid response element (GRE). Often the activation of the GR is associated with a parallel activation of the transcription factor C/EBP-, followed by de novo synthesis of p21(waf1/cip1), which in turn is mediating the GR dependent inhibition of the cell cycle. Both, GR and C/EBP- are necessary to induce and activate p21(waf1/cip1) and to arrest the cell cycle at the transition from the G1- to the S-phase. Fibroblasts and bronchial smooth muscle cells are centrally involved in airway remodeling of patients with asthma and COPD. Therefore three cell types were chosen to assess the effect of the classical and new glucocorticoids on GR activation, cell proliferation and extracellular matrix deposition under different conditions. Four cell culture conditions were chosen to mimic different stages of inflammation: confluent cells without serum representing intact, not damaged tissue; confluent cells with 5% fetal calf serum to mimic early stages of inflammation; sub-confluent cells without serum to represent damaged tissue with no active inflammatory process; sub-confluent cells with 5% serum to represented tissue with active inflammation-induced lung tissue remodeling. The results showed that cell density and inflammation alter the localization and function of the GR. In sub-confluent cells dexamethasone activated the nuclear accumulation and DNA binding of the GR persistently, while in confluent cells its activity declined. In sub-confluent cells, GR interacted with a 42 kDa C/EBP-α isoprotein, which resulted in an up-regulation of p21(Waf1/Cip1) expression and suppression of proliferation. In confluent cells glucocorticoids induced p27(Kip1) expression via p38 MAP kinase and a 52 kDa C/EBP-β isoprotein. Furthermore, p27(Kip1) did not mediate the anti-proliferative effect of glucocorticoids, but simultaneous inhibition of p21(Waf1/Cip1) and p27(Kip1) unlocked contact inhibition in confluent cell cultures. The studies further showed that in lung fibroblasts the long acting steroid mometasone in contrast to ciclesonide or the short acting steroids dexamethasone, budesonide or fluticasone did not induce tachyphylaxis and had a preferable effect on airway remodeling. Mometasone maintained the increased GR activity significantly over 24 hours, compared to that induced by dexamethasone which peaked at 6 hours and declined thereafter. Similarly drug specific effects were observed for the expression of C/EBP- and p21(Waf1/Cip1). The anti-proliferative effect of mometasone was significantly stronger and long lasting compared to all other steroids. Similar anti-proliferative effects were observed in bronchial smooth muscle cells. Regarding airway remodeling we observed that mometasone, in contrast to all other steroids, did not further stimulate serum dependent synthesis and deposition of extracellular matrix and did not affect matrix metalloproteinase-2 and -9 expression or activity. Therefore this novel steroid will have a lower modulatory effect of tissue remodeling compared to the other steroids. Interestingly the long duration effects of the new glucocorticoid ciclesonide originated from the metabolism of the epithelial cells. In conclusion, our findings suggest that the action of glucocorticoids differs under non-inflammatory and inflammatory conditions in human lung fibroblasts and bronchial smooth muscle cells. Different doses of inhaled steroids might therefore be applied in patient with asthma and COPD dependent on the current status of inflammation in order to reduce side effects. The long acting glucocorticoid mometasone might help to reduce overall steroid dosage and might thereby reduce the risk of tachyphylaxis, enhanced tissue remodeling and other typical steroid side effects. Ciclosonide is a new long acting steroid, but its action on mesenchymal cells depends on the function of epithelial cells. These studies not only improve the understanding of the effect of different glucocorticoid on mesenchymal lung cells but might also help to find optional treatment doses.

Within the context of the establishment of a virosomal-based subunit malaria vaccine including synthetic peptides derived from Plasmodium falciparum antigens demonstrated that it is possible to improve immunogenic properties of synthetic peptides in a medicinal chemistry type optimization process. A close to natural conformation of peptides appears to be crucial for the induction of parasite-binding and inhibitory antibodies. In the case of a circumsporozoite protein (CSP) derived synthetic peptide we showed that this is achieved by conformational restriction of the peptide. In a phase Ia clinical trial of the virosomally formulated apical membrane antigen(AMA-1) -derived 49 amino acid long phosphatidylethanolamin peptide conjugate AMA49-CPE, we measured both vaccine induced humoral and cellular responses. In 50% of the volunteers a peripheral blood mononuclear cells (PBMC) lymphoproliferative response specific for AMA49-CPE was elicited. Moreover, all volunteers who developed high titers of parasite cross-reactive IgG in immunofluorescence assay (IFA) and Western blot analysis, were positive in the antisynthetic peptide lymphoproliferation. No interference was found between the magnitude of the pre-existing influenza specific T cell response and the vaccinationinduced AMA49-CPE specific humoral and cellular immune response. We demonstrate that the virosomal antigen delivery platform combined with surfaceanchored synthetic peptides is suitable to elicit specific human CD4 T cell responses. Mycobacterium ulcerans infection (Buruli ulcer) is unique among mycobacteria in that much of the pathology and local immunosuppression appears to be mediated by the production of a cytopathic toxin called mycolactone. IFN-γ ELISpot was used to compare reactivity of PBMC of Buruli ulcer patients with complex antigens such as isopentenyl pyrophosphate (IPP), purified protein derivative (PPD) and influenza antigens prior to and after surgical treatment. The results demonstrated that M. ulcerans infection-associated systemic reduction in IFN-γ response is not confined to stimulation with mycobacterial antigens. Immunosuppression reversed after surgical treatment, indicating that this T cell anergy is not the consequence of a genetic defect but rather related to M. ulcerans infection. In the affected tissue we detected mRNA coding for innate immune system markers, even though the lack of inflammatory responses is a hallmark of Buruli ulcer disease. These were distributed within the lesion in a very focal and heterogeneous way. IL-8, IL-6, and TNF-α mRNA was detectable by real time PCR in all lesions, whereas the expression of IFN-α, IL12p40, IL-10, and IFN-γ was only found in some lesions. Correlations of the distribution of mRNA encoding for the activation marker CD83 and the DC subset markers CD123 and CD11c indicate that both activated plasmacytoid and myeloid DC were present in the lesions. These results suggest that adaptive M. ulcerans specific immune responses may develop once therapeutic interventions have limited the production of mycolactone.

Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. 3D architectures might also play a role in impairing immunorecognition of cancer cells by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens. Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA-) melanoma cell lines on poly-Hydroxyethylmethacrylate-coated plates, resulted in generation of 3D multicellular tumor spheroids (MCTS). Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10-15 days) MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. To obtain an insight into the role played by tumor architecture in the elicitation of specific gene expression patterns, we addressed gene expression profiles of NA8 melanoma cells cultured in two-dimensional monolayers (2D) or in 3D (MCTS). Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with more than threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, more than threefold, upregulation of the expression of >100 transcripts, while 73 transcripts were more than threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-α, -β, and γ), IL-8, CCL20 (MIP-3α), and Angiopoietin-like 4 were significantly upregulated, whereas basic-FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process. Then we investigated the effects of 3D culture on the recognition of melanoma cells by antigenspecific HLA class I-restricted Cytotoxic T-Lymphocytes (CTL). IFN-γ production can be used as a surrogate marker for tumor cell immunorecognition. Co-culture of melanoma spheroids with HLA-A0201 restricted Melan-A/MART-127-35-specific CTL clones resulted in significantly defective TAA recognition by CTL as compared to 2D, as witnessed by decreased IFN-γ production and decreased Fas Ligand, perforin and granzyme B gene expression. Indeed, Melan- A/MART-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D tumor cell cultures. Concomitantly, a parallel decrease of HLA class I molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures. We further identified a multiplicity of mechanisms potentially involved. In particular : 1) MCTS per se limit CTL capacity of recognizing HLA class I restricted antigens by reducing exposed cell surfaces. 2) Expression of melanoma differentiation antigens is down-regulated in tumor cell spheroids as compared to 2D unrelated to hypoxia or increased Oncostatin M gene expression but rather to decreased MITF gene expression. 3) Expression of HLA class I molecules is frequently down-regulated in melanoma MCTS, as compared to 2D, possibly due to decreased IRF-1 gene expression. 4) Lactate production by melanoma cells is increased in MCTS, as compared to 2D and lactate significantly inhibits TAA triggered IFN-γ production by CTL. Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL and a constellation of mechanisms are involved in causing this impairment of immunorecognition.

Lung cancer is the leading cause of cancer-related mortality in the world, whereby Non Small Cell Lung Carcinomas (NSCLC) constitute 80% of all lung tumors. Whereas in Stage I and II NSCLC surgical resection with or without adjuvant chemotherapy currently represents the most frequently applied treatment, in late stage NSCLC, representing 70% of all cases, chemotherapy or radiotherapy are mainly palliative. Thus, the poor prognosis and the limited therapeutic options available urge the development of new approaches. Among these, active specific immunotherapy targeting Cancer/Testis Antigens (CTA) might represent a valuable additional treatment in NSCLC. CTA have been shown to represent promising targets in different types of cancer as they are silent in healthy adult tissues except in testis and placenta. These tissues do not present antigenic epitopes as they are deficient in MHC expression. Moreover, CTA are expressed by various tumors of different histology, stage and grade, and in some tumors, expression has been found to be correlated with poor disease specific survival. In this study first the prevalence and expression patterns of several CTA (MAGE-A1, -A2, -A3, -A4, -A10, -A12 and NY-ESO-1) in freshly excised NSCLC were investigated at gene and protein level. Tumor specimens (12 adeno-, 17 squamous cell and 4 large cell carcinomas) were obtained from HLA-A*0101 and/or HLA-A*0201 positive patients. CTA expression was detected in five adeno-, eight squamous cell and in two large cell carcinoma samples (45.5%). MAGE-A10 and -A12 were the most frequently (10/15 and 12/15 specimens, respectively) and MAGE-A1, -A4 and NY-ESO-1 the least frequently expressed genes (6/15, 6/15 and 4/15 specimens, respectively). In 10/15 positive cases at least four CTA genes were concomitantly expressed. These results at gene level were widely confirmed by protein detection, the few discrepancies being explained by focal CTA expression limited to defined tumor areas. Immune responsiveness towards MAGE-A1 and -A3 (HLA-A*0101 restricted), MAGE-A4, -A10, multi-MAGE-A (an epitope shared by several MAGE-A antigens) and NY-ESO-1 (HLA-A*0201 restricted) epitopes was evaluated in cancer patients to assess whether a specific cellular response could be detected or generated upon ex vivo stimulation. Induction of CTL was performed on expanded CD8+ T lymphocytes infiltrating the tumors (TIL), possibly enriched in activated specific T cells, eventually due to the presence of antigen. After successful expansion, CD8+ cells were repeatedly stimulated with autologous mature IL-4-DC pulsed with CTA peptides and/or infected with a recombinant vaccinia virus (rVV) encoding the corresponding epitopes together with the gene encoding human CD80. These vectors were constructed during the present study to provide highly effective immunogenic reagents with the perspective of possible clinical application. CTA specific CTL response could be observed in 7/26 populations. In six cultures, cytotoxic activity was low and did not correlate with expression of specific CTA in the original tumor specimens. These CTL responses could possibly be attributed to a primary in vitro sensitization. However, in one case stimulation of TIL with rVV infected APC revealed a high level of MAGE-A10 specific CTL response detectable by cytotoxicity assays and multimer staining. The corresponding gene, encoding the target epitope, was highly expressed in the original tumor. In NSCLC, CTA specific CTL sensitization in TIL, as detectable upon repeated stimulation with a panel of well defined peptides and highly effective APC, is rare. On the other hand, strong CTA specific CTL responses could frequently be generated from peripheral blood lymphocyes of healthy donors, upon stimulation of large numbers of effector cells with antigen pulsed DC obtained by GM-CSF/IFNα induction. The concomitant expression of multiple CTA in NSCLC and the possibility of natural CTL responses in these cancers may support the development of specific vaccination protocols using multi antigen vaccine preparations of CTA.

The induction of cytotoxic T lymphocyte (CTL) responses is of high relevance in immunological defense against intracellular pathogens and tumor cells. While humoral immune responses are successfully induced by a number of vaccines, the activation of cellular immune responses has only been addressed more recently. The development of novel immunogens from live attenuated vaccines to subunit vaccines demands efficient and safe adjuvants to improve their immunogenicity. Importantly, there are only three adjuvants licensed for human use: aluminium salts, MF59 (microfluidized detergent stabilized oil in water emulsion) and IRIV (immunopotentiating reconstituted influenza virosomes). Aluminium salts are the most widely used adjuvants and their efficacy in enhancement of humoral responses is well documented. They are ineffective in the induction of cellular responses, whereas IRIV and MF59 might be effective, in addition to humoral responses, also in the induction of cellular responses. The aim of our group, working in the field of cancer immunotherapy, is induction of CTL specific to melanoma associated antigens. The monitoring of a clinical phase I/II trial has demonstrated increased frequencies of specific CTL in peripheral blood upon administration of antigenic epitopes encoded as minigenes with costimulatory molecules in a recombinant vaccinia virus. In the heterologous vaccination protocol adopted, however, high CTL frequencies were not sustained upon administration of the same epitopes as synthetic peptides. This pattern prompted the search for appropriate adjuvants enhancing peptide induced CTL responses. In this thesis work we focused on the in vitro characterization of immune responses elicited by influenza virosomes and on the in vitro evaluation of influenza virosome adjuvance in HLA class I restricted peptide induced CTL responses. We tested empty IRIV admixed with peptides and influenza virosomes encapsulating peptides, both produced by Pevion Biotech Ltd. Due to the low encapsulation efficiency of IRIV per se, the production of the second formulation required encapsulation of peptides into liposomes and subsequent fusion with chimeric IRIV. Thus, we characterised immune responses elicited by empty IRIV and empty chimeric IRIV fused with empty liposomes (FCIRIV). Then, we evaluated their adjuvant capacity by testing CTL induction in the presence of IRIV admixed with peptides and by peptides encapsulated in FCIRIV as compared to CTL induction by peptides in absence of influenza virosomes. For IRIV admixed with peptides we addressed induction of CTL specific for the highly immunogenic Influenza matrix 58-66 (IM58-66) and to the immunodominant melanoma associated Melan-A/ Mart-127-35 HLA-A201 restricted epitopes. For peptides encapsulated in FCIRIV we addressed induction of CTL specific for the L27Melan-A/Mart-126-35 HLAA0201 restricted epitope. Our results demonstrate that all influenza virosome formulations under investigation induce antigen triggered CD4+ T cell proliferation characterized by a T helper 1 cytokine profile. Further dissection of CD4+ T cells identified CD4+CD45RO+ cells as proliferative responders to IRIV stimulation and no major cell proliferation could be induced in cord blood mononuclear cell cultures. These findings indicate that the majority of CD4+ T cells responding to IRIV are antigen experienced. In addition, supernatants of IRIV stimulated PBMC cultures favoured maturation of dendritic cells, as demonstrated by upregulation of HLA-ABC, CD86 and CD83. Both, influenza virosomes admixed with peptides or encapsulating peptides significantly enhanced specific CTL induction, as detected by multimer staining and cytotoxicity assays. CTL induction experiments in presence of irradiated CD4+ T cells indicated that IRIV CTL adjuvance required CD4+ T cell activation. In addition, transwell cultures pointed to a key role of cytokines in IRIV mediated CTL adjuvance. In contrast to empty IRIV, FCIRIV with encapsulated peptides were characterized by CD4+ T cell independent adjuvant potential, possibly attributable to influenza virosome delivery capacities. Taken together, our results demonstrate that influenza virosomes are endowed with the capacity to enhance HLA class I restricted CTL induction in vitro. Importantly, this could be demonstrated not only for the highly immunogenic IM58-66 epitope, but also for the melanoma associated epitopes L27Melan-A/Mart-126-35 and Melan-A/Mart-127-35. Moreover, CTL induced by L27Melan-A/Mart-126-35 encapsulated in FCIRIV were capable of recognizing and lysing tumor cells that constitutively express the Melan-A/Mart-1 antigen. These in vitro findings encourage further evaluation of influenza virosome CTL adjuvance in vivo.

Recombinant poxviruses expressing immuno-modulatory molecules together with specific antigens might represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in clinical trials, that co-expression of costimulatory molecules (CD80 and CD86) could increase the immunogenic capacity of a recombinant Vaccinia virus (rVV) also encoding different tumor associated antigens. In order to further investigate the capacity of these vectors to provide ligands for different co-stimulatory pathways relevant in the generation of CD8+ T cell responses, we designed a recombinant virus expressing CD40 ligand (CD154rVV). This co-receptor, expressed on activated CD4+ T cells, upon binding CD40 expressed on antigen presenting cells (APC) has been reported to increase their antigen presentation and immunomodulatory capacities. To investigate the potency of CD154rVV in CTL generation, different types of infection were performed in cultures containing APC and CD8+ T cells. Phenotypic characterization of infected iDC showed that CD154rVV enhances their activation and maturation, measured by increased expression of CD83 and CD86, as compared to wild type Vaccinia virus as control. Cytokine gene expression was evaluated by quantitative real time PCR. As expected, control virus infection triggered cytokine gene expression in APC and T-cells. However, typical APC cytokines such as GM-CSF, TNF-α and IL-15 and, on the other hand, typical T cells cytokines such as IL-2 and IFN-γ seemed to be expressed to a higher extent in CD154rVV infected cultures. Furthermore, as a landmark of the CD40/CD154 pathway, IL-12p40 gene transcription in APC was exclusively induced by CD154rVV infection. The latter factor is also known to play a major role in CTL priming. Complementary, as expected, control virus infection triggered IL-6 gene expression, which is known to render tumor specific T cells refractory to T regulatory cell activity. Nevertheless, this expression could not be further enhanced by CD154rVV. Activation of specific CD8+ T cells was investigated by using the MART-1/Melan-A27-35 epitope as model antigen and monitored by tetramer staining and cytotoxicity assays. We found that increased numbers of specific cytotoxic CD8+ T cells were induced by the specific peptide in the presence of the CD154rVV activated APCs, as compared to control virus. Finally, CD154rVV was demonstrated to enhance T cell proliferation, mainly CD4+ T cell, in culture of infected APCs with autologous T cells. Taken together, these data indicate that functional CD154 expression from recombinant Vaccinia virus infected cells induces APC activation and maturation, thereby enhancing antigen specific CD8+ T cell generation. Such recombinant vector might help bypassing the requirement for activated helper cells, thus qualifying as a potentially relevant reagent in the generation of CD8+ T cell responses in cancer immunotherapy.