Silvina Gazzaniga

Dendritic cell-based vaccination in cancer: therapeutic implications emerging from murine models

Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment.

Vaccination with dendritic cells charged with apoptotic/necrotic B16 melanoma induces the formation of subcutaneous lymphoid tissue.

Antigen presentation by dendritic cells (DC) is of key importance for the initiation of the primary immune response. Mice vaccinated with DC charged with apoptotic/necrotic B16 cells (DC-Apo/Nec) are protected against B16 challenge. The aim of this study was to assess vaccine cell migration in our system and to find out if there is an immunological response taking place at the vaccination site. The formation of a pseudocapsule, peripheral node addresin expression in small venules, and the recruitment of a wide variety of cellular populations, including macrophages, polymorphonuclear lymphocytes, and CD8+ and CD4+ T lymphocytes found in association with DC, evidenced the formation of tertiary lymphoid tissue in the vaccination site in our experimental system.

ISOLATION AND MOLECULAR CHARACTERIZATION OF A MOUSE RENAL MICROVASCULAR ENDOTHELIAL CELL LINE

SummaryMany of the morphological defects associated with embryonic alcohol exposure are a result of cell death. During limb development, ethanol administration produces cell death in the limb and digital defects, including postaxial ectrodactyly. Because an accumulation of reactive oxygen species (ROS) is produced in adult and embryonic tissues by ethanol exposure, this investigation examines the possibility that ethanol-induced cell death in the limb is a result of ROS. Using an in vitro primary culture of limb mesenchyme, the effects of hydrogen peroxide (H2O2) and ethanol on cell death and differentiation were examined. In addition, a dichlorofluorescein diacetate assay was performed to determine the relative intracellular ROS levels after exposure to several concentrations of ethanol and H2O2. Exposure of 1 to 100 μM H2O2 resulted in a 1.08–1.21 times control increase in cartilage matrix accumulation. Cell death was increased 1.69–2.76 times the untreated control value. Production of ROS ranged from 1.25–1.51 times untreated controls. Ethanol exposure of 0.25 to 1.00% (v/v) did not affect cartilage matrix accumulation but resulted in an increase of cell death (1.45–2.31 lower than that produced by 1 μM H2O2. On the basis of the correlation between ROS level produced by H2O2, it was concluded that ethanol-induced cell death in limb mesenchyme is a result of a non-ROS-mediated mechanism. Therefore, in addition to ethanol-induced cell death mediated by ROS reported in the literature, ethanol-induced cell death can be induced in limb mesenchyme by mechanisms that are not dependent upon ROS.Abstract Murine endothelial cells (ECs) have proven difficult to obtain and maintain in culture. Long-term maintenance of normal ECs remains a difficult task. In this article we report the establishment of the first cellular line of renal microvascular endothelium obtained from normal tissue. Cells were isolated, cloned, and maintained by serial passages for longer than 24 mo, using endothelial cell growth supplement (ECGS) and gelatin-coated plates. Their morphology and ultrastructure, expression of von Willebrand factor, presence of smooth muscle α-actin, vimentin, cytokeratin filaments, capillary structures formed on Matrigel, and some typical ECs surface molecules were the criteria used to characterize cultured ECs. When examined for responsiveness to Shiga toxin-1, 13–20% of cytotoxicity was observed when coincubated with lipopolysaccharides. This cytotoxicity was not observed for normal lung ECs (1G11). Consequently, REC-A4 line retains characteristics of resting microvascular ECs and represents a useful in vitro model to study biological and physiopathological properties of renal endothelium.

Atheroprotective remodelling of vascular dermatan sulphate proteoglycans in response to hypercholesterolaemia in a rat model.

Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high‐cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal‐to‐media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo‐intima rearrangement were observed in high‐cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high‐cholesterol animals compared with control (P < 0.01 and P < 0.05, respectively). Versican was increased in high‐cholesterol animals (P < 0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high‐cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6‐sulphate to 4‐sulphate disaccharides was increased in high‐cholesterol animals (P < 0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.

Anti-melanoma vaccinal capacity of CD11c-positive and -negative cell populations present in GM-CSF cultures derived from murine bone marrow precursors

We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC-) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC- fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC- fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccines ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo.

Altered expression of cytokines in mice infected intranasally with two syncytial variants of Herpes simplex virus type 1.

Immune evasion strategies are important for the onset and the maintenance of viral infections. Many viruses have evolved mechanisms to counteract or suppress the host immune response. We have previously characterized two syncytial (syn) variants of Herpes simplex 1 (HSV-1) strain F, syn14-1 and syn17-2, obtained by selective pressure with a natural carrageenan. These variants showed a differential pathology in vaginal and respiratory mucosa infection in comparison with parental strain. In this paper, we evaluated the modulation of immune response in respiratory mucosa by these HSV-1 variants. We observed altered levels of Tumor Necrosis Factor-α and Interleukin-6 in lungs of animals infected with the syn14-1 and syn17-2 variants compared with the parental strain. Also, we detected differences in the recruitment of immune cells to the lung in syn variants infected mice. Both variants exhibit one point mutation in the sequence of the gene of glycoprotein D detected in the ectodomain of syn14-1 and the cytoplasmic tail of syn17-2. Results obtained in the present study contribute to the characterization of HSV-1 syn variants and the participation of the cellular inflammatory response in viral pathogenesis.

CD207⁺ cells recruitment to the vaccination site and draining lymph nodes after the administration of DC-Apo/Nec vaccine in mice.

De novo ectopic lymphoid tissue formation is known to occur in certain disease and inflammatory settings. After an effective vaccination with dendritic cells (DC) charged with melanoma apoptotic/necrotic cells (Apo/Nec), a subcutaneous tertiary lymphoid structure was organized, where retained vaccine cells interacted with recruited inflammatory and T cells. In this work we report for the first time the recruitment of two morphologically different CD207(+) cells to vaccination site. The time-course behavior of CD207(+) cells was reciprocal between vaccination site and draining lymph nodes (DLNs). After 6-10 days, CD207(+) cells localized at the paracortical region of DLNs, in close contact with T cell population. DLNs were enriched in a peculiar MHCII(+) CD11c((-)) CD207(+) population, whose role remains to be determined. Whether CD207(+) cells migration to the vaccination site can be associated with a differential anti-tumoral response remains as an open and exciting question.

Selective hemangioma cell dysfunction and apoptosis triggered by in vitro treatment with imiquimod

Infantile hemangiomas are the most common benign tumors of infancy, characterized by unregulated angiogenesis and endothelial cells with high mitotic rate. Although spontaneous regression occurs, sometimes treatment is required and alternatives to corticosteroids should be considered to reduce side effects. Imiquimod is an imidazoquinoline, approved for some skin pathologies other than hemangioma. It is proposed that the effectiveness of imiquimod comes from the activation of immune cells at tumor microenvironment. However, the possibility to selectively kill different cell types and to directly impede angiogenesis has been scarcely explored in vitro for endothelial cells. In this work we showed a dramatic cytotoxicity on hemangioma cell, with a significant lower IC50 value in hemangioma compared to normal endothelial cells and melanoma (employed as a non-endothelial tumor cell line). Nuclear morphometric and flow-cytometry assays revealed imiquimod-induced apoptosis on hemangioma and melanoma cells but a small percentage of senescence on normal endothelial cells. At sub-lethal conditions, cell migration, a key step in angiogenesis turned out to be inhibited in a tumor-selective manner along with actin cytoskeleton disorganization on hemangioma cells. Altogether, these findings pointed out the selective cytotoxic effects of imiquimod on transformed endothelial cells, evidencing the potential for imiquimod to be a therapeutic alternative to reduce extensive superficial hemangioma lesions.