Aurelia Rughetti

Tumor-Associated Tn-MUC1 Glycoform Is Internalized through the Macrophage Galactose-Type C-Type Lectin and Delivered to the HLA Class I and II Compartments in Dendritic Cells

The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcalpha1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines.

Recombinant tumor-associated MUC1 glycoprotein impairs the differentiation and function of dendritic cells

Tumors exploit several strategies to evade immune recognition, including the production of a large number of immunosuppressive factors, which leads to reduced numbers and impaired functions of dendritic cells (DCs) in the vicinity of tumors. We have investigated whether a mucin released by tumor cells could be involved in causing these immunomodulating effects on DCs. We used a recombinant purified form of the MUC1 glycoprotein, an epithelial associated mucin that is overexpressed, aberrantly glycosylated, and shed during cancer transformation. The O-glycosylation profile of the recombinant MUC1 glycoprotein (ST-MUC1) resembled that expressed by epithelial tumors in vivo, consisting of large numbers of sialylated core 1 (sialyl-T, ST) oligosaccharides. When cultured in the presence of ST-MUC1, human monocyte-derived DCs displayed a modified phenotype with decreased expression of costimulatory molecules (CD86, CD40), Ag-presenting molecules (DR and CD1d), and differentiation markers (CD83). In contrast, markers associated with an immature phenotype, CD1a and CD206 (mannose receptor), were increased. This effect was already evident at day 4 of DC culture and was dose dependent. The modified phenotype of DCs corresponded to an altered balance in IL-12/IL-10 cytokine production, with DC expressing an IL-10highIL-12low phenotype after exposure to ST-MUC1. These DCs were defective in their ability to induce immune responses in both allogeneic and autologous settings, as detected in proliferation and ELISPOT assays. The altered DC differentiation and Ag presentation function induced by the soluble sialylated tumor-associated mucin may represent a mechanism by which epithelial tumors can escape immunosurveillance.

Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation

Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen‐associated molecular patterns. In particular, C‐type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca2+‐dependent manner. Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC‐based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C‐lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for α‐N‐acetylgalactosamine (GalNAc or Tn)‐carrying tumor‐associated antigens to improve DC performance. MGL expressed by ex vivo‐generated iDCs from healthy donors was engaged by a 60‐mer MUC19Tn‐glycopeptide as a Tn‐carrying tumor‐associated antigen, and an anti‐MGL antibody, as a specific MGL binder. We demonstrated that MGL engagement induced homotrimers and homodimers, triggering the phosphorylation of extracellular signal‐regulated kinase 1,2 (ERK1,2) and nuclear factor‐κB activation. Analysis of DC phenotype and function demonstrated that MGL engagement improved DC performance as antigen‐presenting cells, promoting the upregulation of maturation markers, a decrease in phagocytosis, an enhancement of motility, and most importantly an increase in antigen‐specific CD8+ T‐cell activation. These results demonstrate that the targeting of MGL receptor on human DCs has an adjuvant effect and that this strategy can be used to design novel anticancer vaccines.

Transfected human dendritic cells to induce antitumor immunity

Dendritic cells are professional antigen-presenting cells able to prime naive T lymphocytes and regulate steadily the delicate balance between tolerance and activation during the immune response. In past years several reports have shown that genetically engineered dendritic cells (DCs) can be a powerful tool for inducing an antigen-specific immune response. The use of such modified antigen-presenting cells is a real working hypothesis in preclinical studies and in clinical vaccination approaches for cancer treatment. The definition of optimal transfection conditions for preserving DC survival and functionality is necessary to design a correct immunotherapeutic protocol. Different lipid-based transfection compounds were studied for their effects on DC survival, phenotype and functional properties. All the transfection procedures were able to select DCs with a higher expression of activation and costimulatory molecules (ie MHCII-DR, CD83, CD86, CD25) than the untreated DCs. However, only two compounds (LipofectAMINE PLUS and FuGENE 6), preserved or even increased the immunopotency of DCs as antigen-presenting cells. These protocols were applied to modify DCs in order to express an epithelial tumor-associated antigen, MUC1, and such cells were able to induce in vitro a specific immune response in healthy donors.

MAGE-A and NY-ESO-1 expression in cervical cancer: Prognostic factors and effects of chemotherapy

OBJECTIVE The aim of this study was to evaluate the prevalence of cancer testis tumor-associated antigens MAGE-A and NY-ESO-1 in cervical cancer and correlate expression patterns with clinicopathologic parameters and prognosis. STUDY DESIGN One hundred sixty-two cervical cancer samples from 109 patients who were treated with radical hysterectomy, neoadjuvant chemotherapy, or pelvic disease recurrence were analyzed by immunohistochemistry. RESULTS MAGE-A was expressed by 32/94 (34%) and 7/15 (47%) previously untreated and recurrent tumors, respectively. NY-ESO-1 was expressed by 46/94 (49%) and 6/15 (40%) previously untreated and recurrent tumors, respectively. MAGE-A in early stage tumors was correlated to tumor size and lymph node metastases (P = .024 and P = .046, respectively) whereas NY-ESO-1 to tumor grading (P = .039). CONCLUSION Cervical cancer frequently expresses cancer testis tumor-associated antigens. MAGE-A and NY-ESO-1 expression rates are not influenced by systemic therapies. Cancer testis tumor-associated antigens are correlated to common prognostic factors.

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4+CD25+FOXP3+ circulating cells (CD4 Treg). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4+ Treg cells rapidly decreased after primary tumour debulking, while CD8+CD25+FOXP3+ (CD8 Treg) cells are not detectable in peripheral blood. Similar results on CD4 Treg were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 Treg cells are both present in neoplastic tissue. Interleukin (IL)‐10 serum levels decrease after surgery, while no changes are observed in transforming growth factor‐β1 and IL‐6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8+ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating Treg and an increase in CD8 T‐cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.

Isolation of MUC1-primed B lymphocytes from tumour-draining lymph nodes by immunomagnetic beads

Abstract The humoral immune response against a tumour-associated antigen, polymorphic epithelial mucin (PEM, MUC1) in cancer patients was studied by isolating specific B cells primed for the antigen. Human B lymphocytes from tumour-draining lymph nodes, obtained from 12 patients with epithelial cancers, were immunoselected with magnetic beads coated with a 60mer synthetic peptide corresponding to three tandem repeats of the protein core of the MUC1 antigen. Short-term cultures of B cells were established utilizing interleukin-10 (IL-10), IL-4 and monoclonal antibody anti-CD40, and were maintained for a maximum of 3␣weeks. B cell culture supernatants contained human anti-MUC1 antibodies, as detected by enzyme-linked immunosorbent assay, in 6/12 of the patients tested. Five of these patients, all with early-stage cancer, also had high levels of circulating anti-MUC1 IgM antibodies in the serum. A significant correlation was found (two-tailed P = 0.041) between the presence of circulating anti-MUC1 antibodies and the ability to isolate PEM-specific B cells from tumour-draining lymph nodes. The technique proposed provides a useful method for the analysis of natural immunity against defined tumour antigens.

Monoclonal Antibodies in Gynecological Cancer: A Critical Point of View

During the last decades, several improvements in treating gynecological malignancies have been achieved. In particular, target therapies, mostly monoclonal antibodies, have emerged as an attractive option for the treatment of these malignancies. In fact, various molecular-targeted agents have been developed for a variety of malignancies with the objective to interfere with a precise tumor associated receptor, essential for cancer cell survival or proliferation, blocking its function, of the cancer cells. Alternatively, monoclonal antibodies have been developed to block immune suppression or enhance functions of immune effector cells. So far, several monoclonal antibodies have been tested for clinical efficacy for the treatment of gynecological cancers. Antibodies against Vascular Endothelial Growth Factor (VEGF) and Epidermal Growth Factor Receptor (EGFR) have been used in different neoplasms such as ovarian and cervical cancer. Catumazumab, a bivalent antibody against CD3 and EpCAM, is effective in the treatment of neoplastic ascites. Other antibodies are peculiar for specific cancer-associated antigen such as Oregovomab against CA125 or Farletuzumab against the folate receptor. Here we describe the preclinical and clinical experience gained up to now with monoclonal antibodies in tumors of the female genital tract and trace future therapeutic and research venues.

Regulated expression of MUC1 epithelial antigen in erythropoiesis

Summary. MUC1 is a large surface glycoprotein expressed by epithelial cells, which is overexpressed and aberrantly glycosylated in carcinomas. MUC1 is involved in epithelial cell interactions and appears to function as a signal‐transducing molecule. The finding that MUC1 can also be expressed in the haematopoietic lineages prompted us to further investigate the possible function(s) of this molecule in haematopoietic cells. In bone marrow differentiating cells, MUC1 was strongly and selectively expressed during erythropoiesis; it was also weakly expressed during megakaryocytopoiesis and granulomonocytopoiesis; however, no correlation between MUC1 and differentiation marker expression was observed in these lineages. In vitro CD34+ cells, induced towards erythroid differentiation, acquired MUC1 transiently, while expressing increasing levels of the lineage marker glycophorin A. MUC1 was absent in the circulating erythrocytes. During erythropoiesis, MUC1 expression was transcriptionally regulated and the molecule underwent phosphorylation. To investigate the possible role of MUC1 during erythropoiesis, we studied the ability of MUC1 to act as ligand for cell–cell interaction. The sialylated MUC1 glycoforms selectively expressed on erythroid cells were able to bind the macrophage‐restricted molecule sialoadhesin. These results suggest that MUC1 can function as a cross‐talk molecule between the erythroblasts and the surrounding cells during erythropoiesis.

Human antibodies against the polymorphic epithelial mucin in ovarian cancer patients recognise a novel sequence in the tandem repeat region

The humoral immune response to the polymorphic epithelial mucin (PEM) was studied by characterising the reactivity of human antibodies generated by EBV-immortalised B-cells from tumour-draining lymph nodes of ovarian cancer patients. All the human antibodies, selected in ELISA for their reactivity to the protein tandem core repeat sequence, reacted with PEM-expressing tumour cells. Aberrant glycosylation of the peptide core of the PEM molecule in cancer cells leads to the exposure of peptide epitopes that can be considered tumour specific. The epitope mapping of six human antibodies revealed that only one of them contained the PDTR sequence, shown to be the immunodominant epitope in the mouse. Four of the six human antibodies recognised a novel common immunogenic sequence (APPAH) in the tandem repeats. The binding of these human antibodies did not appear to be modulated by the length of the carbohydrate side chains, as shown by O-glycosylation inhibition studies. These results indicate that distinct sequences within the tandem repeat of PEM are target for a humoral immune response in humans. The presence of antibodies directed against different epitopes within the same antigenic region may modulate the antigen presentation process and the ongoing immune response. This data may help in clarifying the mechanisms of the immune response to PEM in cancer patients for the development of PEM-based immunotherapy.