Silvano Ferrini

IL-21 Induces Tumor Rejection by Specific CTL and IFN-γ-Dependent CXC Chemokines in Syngeneic Mice

IL-21 is an immune-stimulatory four α helix cytokine produced by activated T cells. To study the in vivo antitumor activities of IL-21, TS/A murine mammary adenocarcinoma cells were genetically modified to secrete IL-21 (TS/A-IL-21). These cells developed small tumors that were subsequently rejected by 90% of s.c. injected syngeneic mice. Five days after injection, TS/A-IL-21 tumors showed numerous infiltrating granulocytes, NK cells, and to a lesser extent CD8+ T cells, along with the expression of TNF-α, IFN-γ, and endothelial adhesion molecules ICAM-1 and VCAM-1. At day 7, CD8+ and CD4+ T cells increased together with IFN-γ, and the CXC chemokines IFN-γ-inducible protein 10, monokine induced by IFN-γ, and IFN-inducible T cell α-chemoattractant. The TS/A-IL-21 tumor displayed a disrupted vascular network with abortive sprouting and signs of endothelial cell damage. In vivo depletion experiments by specific Abs showed that rejection of TS/A-IL-21 cells required CD8+ T lymphocytes and granulocytes. When injected in IFN-γ-deficient mice, TS/A-IL-21 cells formed tumors that regressed in only 29% of animals, indicating a role for IFN-γ in IL-21-mediated antitumor response, but also the existence of IFN-γ-independent effects. Most immunocompetent mice rejecting TS/A-IL-21 cells developed protective immunity against TS/A-pc (75%) and against the antigenically related C26 colon carcinoma cells (61%), as indicated by rechallenge experiments. A specific CTL response against the gp70-env protein of an endogenous murine retrovirus coexpressed by TS/A and C26 cells was detected in mice rejecting TS/A-IL-21 cells. These data suggest that IL-21 represents a suitable adjuvant in inducing specific CTL responses.

Gene therapy of glioblastoma multiforme via combined expression of suicide and cytokine genes: a pilot study in humans

Retrovirus-mediated gene therapy is a particularly attractive approach for glioblastoma multiforme (GBM), given the poor prognosis of this tumour and its localized proliferation in post-mitotic tissue. In this study we assessed, for the first time in humans, the therapeutic potential of a newly designed bicistronic Moloney vector (pLIL-2-TK), combining the expression of a suicide gene (thymidine kinase, tk) with an immunomodulatory gene (human interleukin 2, IL-2). Evidence of transgene activity in the treated tumours is presented.

Identification of a novel domain of HIV tat involved in monocyte chemotaxis.

Human immunodeficiency virus (HIV) Tat is chemotactic for monocytes and dendritic cells, an activity that could play a key role in the expansion of HIV infection of accessory cells. To date, domains of Tat previously found to interact with cell surface molecules have shown only partial chemotactic activity toward monocytes. Using overlapping Tat peptides, we identify a novel region of Tat with a potent chemotactic activity for monocytes, reaching levels equal to Tat itself. This peptide also provokes monocyte polarization similar to Tat and is able to compete with Tat for induction of monocyte migration. Specific high affinity (k d = 3 × 10−9 m) cell surface binding sites on monocyte cell surfaces for this region of Tat are demonstrated. These data indicate that the majority of Tat effects on monocytes are mediated by a novel region in the cysteine-rich and core domains. These domains are highly conserved among different HIV isolates, suggesting an important role in the establishment of HIV infection.

CD25 + Regulatory T Cell Depletion Augments Immunotherapy of Micrometastases by an IL-21-Secreting Cellular Vaccine

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R γ-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-γ in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-γ production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25− lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-γ. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.

Cancer Stem Cells: A New Paradigm for Understanding Tumor Growth and Progression and Drug Resistance

Normal somatic stem cells (SC) are naturally resistant to chemotherapeutic agents due to their expression of various membrane transporter molecules (such as MDR-1), detoxifying enzymes and DNA repair proteins. In addition, they also have a slow rate of cell turnover and therefore escape from chemotherapeutic agents that target rapidly replicating cells. Cancer stem cells (CSC), being the mutated counterparts of normal SC, also have similar properties, which allow them to survive therapy. These surviving CSC then repopulate the tumor, causing relapse. The purpose of this review is to understand the most current research into the cellular and molecular biology of CSC. Topics that will be explored are the origin of CSC, the CSC niche, the regulation of self-renewal in normal and cancer SC, and CSC as therapeutic targets.

Immunoprevention of HER-2/neu Transgenic Mammary Carcinoma through an Interleukin 12-Engineered Allogeneic Cell Vaccine

This study evaluated the ability of cytokine-engineered allogeneic (H-2q) HER-2/neu-positive cells to prevent tumor development in mammary cancer-prone virgin female BALB/c (H-2d) mice transgenic for the transforming rat HER-2/neu oncogene (BALB-neuT mice). Repeated vaccinations with cells engineered to release interleukin (IL)-2, IL-12, IL-15, or IFN-γ showed that IL-12-engineered cell vaccines had the most powerful immunopreventive activity, with >80% of 1-year-old BALB-neuT mice free of tumors. On the contrary all of the untreated mice and all of the mice vaccinated with IL-12-engineered cells lacking either HER-2/neu or allogeneic antigens developed mammary carcinomas within 22 or 33 weeks, respectively. Whole mount, histology, immunohistochemistry, and gene expression profile analysis showed that vaccination with IL-12-engineered cells maintained 26-week mammary glands free of neoplastic growth, with a gene expression profile that clustered with that of untreated preneoplastic glands. The IL-12-engineered cell vaccine elicited a high production of IFN-γ and IL-4 and a strong anti-HER-2/neu antibody response. Immune protection was lost or markedly impaired in BALB-neuT mice lacking IFN-γ or antibody production, respectively. The protection afforded by the IL-12-engineered cell vaccine was equal to that provided by the systemic administration of recombinant IL-12 in combination with HER-2/neu H-2q cell vaccine. However, IL-12-engineered cell vaccine induced much lower circulating IL-12 and IFN-γ, and therefore lower potential side effects and systemic toxicity.

Subcellular Localization of Activated Leukocyte Cell Adhesion Molecule Is a Molecular Predictor of Survival in Ovarian Carcinoma Patients

Purpose: Currently available clinicopathologic prognostic factors are imperfect predictors of clinical course in advanced-stage epithelial ovarian cancer patients. New molecular predictors are needed to identify patients with higher risk of relapse or death from disease. In a retrospective study, we investigated the prognostic impact of activated leukocyte cell adhesion molecule (ALCAM) expression in epithelial ovarian cancer. Experimental Design: We analyzed the effect of cell-anchorage loss on ALCAM cellular localization in vitro and assessed ALCAM expression by immunohistochemistry in a series of 109 well-characterized epithelial ovarian cancer patient samples. Chi-square test, Kaplan-Meier method, and Cox proportional hazard analyses were used to relate ALCAM cellular localization to clinical-pathologic parameters and to overall survival (OS) rate. Results: Loss of epithelial ovarian cancer cell anchorage was associated both in vitro and in vivo with decreased ALCAM membrane expression. In vivo, ALCAM was localized to cell membrane in normal surface ovarian epithelium, whereas in 67% of the epithelial ovarian cancer samples, membrane localization was decreased or even lost, and the molecule was mainly expressed in cytoplasm. Median OS in this group of patients was 58 months, whereas a median OS was not yet reached in patients with ALCAM membrane localization (P = 0.036, hazard ratio [HR] = 2.0, 95% confidence interval [CI] 1.1 to 3.5). In a multivariate Cox regression model including all the available clinicopathologic variables, loss of ALCAM membrane expression was an independent factor of unfavorable prognosis (P = 0.042, HR = 2.15, 95% CI: 1.0 to 4.5). Conclusions: Decreased/lost ALCAM membrane expression is a marker of poorer outcome in epithelial ovarian cancer patients and might help to identify patients who could benefit from more frequent follow-up or alternative therapeutic modalities.

The ALCAM shedding by the metalloprotease ADAM17/TACE is involved in motility of ovarian carcinoma cells.

Previous findings indicated that the activated leukocyte cell adhesion molecule (ALCAM) is expressed by tumors and plays a role in tumor biology. In this study, we show that ALCAM is shed from epithelial ovarian cancer (EOC) cells in vitro, leading to the generation of a soluble ALCAM (sALCAM), consisting of most of the extracellular domain. A similar sALCAM molecule was also found in the ascitic fluids and sera from EOC patients, suggesting that this process also occurs in vivo. sALCAM is constitutively produced by EOC cells, and this process can be enhanced by cell treatment with pervanadate, phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor (EGF), a known growth factor for EOC. Pharmacologic inhibitors of matrix metalloproteinases (MMP) and of a disintegrin and metalloproteases (ADAM), and the tissue inhibitor of metalloproteinase-3, significantly inhibited sALCAM release by EOC cells. The ADAM17/TACE molecule was expressed in EOC cell lines and ADAM17/TACE silencing by specific small interfering RNA–reduced ALCAM shedding. In addition, inhibitors of ADAM function blocked EOC cell motility in a wound-healing assay. Conversely, a recombinant antibody blocking ALCAM adhesive functions and inducing ALCAM internalization enhanced EOC cell motility. Altogether, our data suggest that the disruption of ALCAM-mediated adhesion is a relevant step in EOC motility, and ADAM17/TACE takes part in this process, which may be relevant to EOC invasive potential. (Mol Cancer Res 2007;5(12):1246–53)

IL-8 induces exocytosis of arginase 1 by neutrophil polymorphonuclears in nonsmall cell lung cancer.

Arginase 1 (ARG1) inhibits T‐cell proliferation by degrading extracellular arginine, which results in decreased responsiveness of T cells to CD3/TCR stimulation. In humans, ARG1 is stored in inactive form within granules of polymorphonuclear neutrophils (PMNs) and gets activated on release. We studied the role of PMNs‐related ARG1 activity in nonsmall cell lung cancer (NSLC), in which tumor‐infiltrating lymphocytes showed reduced proliferation in response to CD3/TCR triggering. Patients with NSCLC had increased ARG1 plasma levels as compared to healthy controls. Furthermore, immunohistochemistry showed that tumor‐infiltrating PMNs display reduced intracellular ARG1, in comparison to intravascular or peritumoral PMNs, suggesting a role of tumor microenvironment in ARG1 release. Indeed, supernatants of NSCLC cell lines induced exocytosis of ARG1 from PMNs. All (4/4) NSCLC cell lines and all (7/7) CD14− cell samples from NSCLC expressed interleukin (IL)‐8 mRNA, whereas TNFα mRNA was expressed by 1 cell line and by 2 tumor specimens. Furthermore, all NSCLC cell lines secreted immunoreactive IL‐8, albeit at different levels. IL‐8 was as effective as TNFα in triggering ARG1 release and the 2 cytokines acted synergistically. Secreted ARG1 was biologically active and catabolized extracellular arginine. The supernatant of IL‐8 gene‐silenced NSCLC cells did not mediate ARG1 release by PMNs. Altogether these findings demonstrate a role of IL‐8 in ARG1 exocytosis by PMNs and indicate that, due at least in part to IL‐8 secreted by NSCLC cells, PMNs infiltrating NSCLC release ARG1. This phenomenon could contribute to local immune suppression.

Differential intracellular trafficking, secretion and endosomal localization of two IL-15 isoforms

To analyze the intracellular trafficking of two IL‐15 isoforms bearing 48‐ or 21‐amino acid leader peptides (L), we have generated cDNA encoding the two proteins fused at the C terminus with green fluorescent protein (GFP). Confocal microscopy analyses showed that, when transfected in CHO cells, 48L IL‐15/GFP was localized in the Golgi apparatus and in early endosomes, while 21L IL‐15/GFP was detectable only in the cytosol. The presence of 48L IL‐15/GFP in endosomes was confirmed by enzyme‐linked immunosorbent assay on endosome‐enriched subcellular fractions. Exogenous IL‐15 was bound and taken up in endosomes by untransfected CHO cells, indicating that endosomal localization was, at least in part, related to a receptor‐mediated uptake. The 48L IL‐15/GFP fusion protein was efficiently secreted by COS‐7 or CHO cell transfectants, while IL‐15 secretion was less efficient in transfectants expressing 21L IL‐15/GFP or untagged 48L or 21L IL‐15. Treatment with brefeldin A or with inhibitors of N‐linked glycosylation further indicated that the 48L IL‐15/GFP is secreted through the endoplasmic reticulum/Golgi pathway. Our data suggest a different trafficking of the two IL‐15 isoforms and multiple mechanisms controlling IL‐15 secretion.