The Sensitizers Nickel Sulfate and 2,4-dinitrofluorobenzene Increase CD40 and IL-12 Receptor Expression in a Fetal Skin Dendritic Cell Line

Dendritic cells (DCs) are antigen-presenting cells (APCs) capable of capturing haptens and to process and present them to T lymphocytes. In order to sensitize T cells for contact hypersensitivity (CHS), skin DCs suffer a maturation process with modifications on their surface molecules. The aim of this work was to evaluate changes induced by two contact sensitizers, 2,4-dinitrofluorobenzene (DNFB) and nickel sulfate (NiSO₄), and a non-sensitizer 2,4-dichloronitrobenzene (DCNB), on the protein levels of two activation markers, CD40 and IL-12 receptor (IL-12R), in a mouse skin dendritic cell line (FSDC). The expression of CD40 and IL-12R proteins was evaluated by western blot assay and direct immunofluorescence microscopy. The results showed that CD40 and IL-12R expression increased significantly after cell exposure to NiSO₄ and DNFB, although DNFB exhibited a stronger activity. There was no effect with DCNB. The epidermal cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF), also used in the experiments, slightly increased the expression of both CD40 and IL-12R and when tested together with the sensitizers the effect was partially additive. The results suggest that the sensitizers DNFB and NiSO₄ are directly involved on the changes of the surface markers CD40 and IL-12R in skin DCs, during the sensitization phase of CHS, and this effect may be enhanced by GM-CSF. In contrast, no effect was observed with DCNB.     

Adhesive interactions between CD34<Superscript>+</Superscript>-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34⁺-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34⁺ progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF- and hSCF for 5 days. Immature CD83^– CD86^low dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1) dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2) dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3) microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34⁺-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.     

Dendritic cells can be rapidly expanded ex vivo and safely administered in patients with metastatic breast cancer

Purpose Immunotherapy using either dendritic cells (DCs) or expanded cytotoxic T cells (CTLs) has received increased interest in the treatment of specific malignancies including metastatic breast cancer (MBC). DCs can be generated ex vivo from monocytes or CD34⁺ precursors. The ability to expand and safely administer CD34-derived DCs in patients with MBC that have received prior cytotoxic chemotherapy has not been evaluated.Methods We enrolled ten patients with MBC that had received prior chemotherapy for the treatment of metastatic disease on a phase I/II trial designed to test the safety and feasibility of administering ex vivo expanded DCs from CD34⁺ progenitor cells.Results Using a cocktail of multiple different cytokines, we could expand DCs 19-fold compared to the initial CD34-selected product, which allowed the administration of as many as six vaccine treatments per patient. Patients received three to six injections i.v. of DCs pulsed with either the wild type GP2 epitope from the HER-2/neu protein or an altered peptide ligand, isoleucine to leucine (I2L). Toxicity was mild, with no patients demonstrating grade III toxicity during the treatment. Two patients with subcutaneous disease had a partial response to therapy, while IFN--producing CD8⁺ T cells could be found in two other patients during treatment.Conclusions This approach is safe and effective in generating a significant quantity of DCs from CD34-precursors.Supported in part by Grants CA 58223 and 89961 from the National Cancer Institute, the Breast Cancer Research Foundation, and RR0046 from the General Clinical Research Center program of the Division of Research Resources, National Institutes of Health.     

α-Tocopheryl succinate sensitizes established tumors to vaccination with nonmatured dendritic cells

Purpose: Dendritic cells (DCs) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors. Methods: In this study we have employed -tocopheryl succinate (-TOS), a nontoxic esterified analogue of vitamin E, as an adjuvant to enhance the effectiveness of DC vaccines in treating established murine Lewis lung (3LL) carcinomas. Results: We demonstrate that locally or systemically administered -TOS in combination with nonmatured DCs injected intratumorally (i.t.) or subcutaneously (s.c.) significantly inhibits the growth of preestablished 10-day tumors (mean tumor volume of 77.5 ± 17.8 mm³ on day 30 post–tumor injection) as compared to -TOS alone (mean tumor volume of 471 ± 68 mm³ on day 30 post–tumor injection). Additionally, the adjuvant effect of -TOS was superior to that of cyclophosphamide (CTX). The mean tumor volume on day 28 post–tumor injection in mice treated with CTX+DCs was 611 ± 94 mm³ as compared to 105 ± 36 mm³ in mice treated with -TOS+DCs. Analysis of purified T lymphocytes from mice treated with -TOS+DC revealed significantly increased secretion of IFN- as compared to T cells from the various control groups. Conclusion: This study demonstrates the potential usefulness of -tocopheryl succinate, an agent nontoxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating established tumors.Abbreviations AO acridine orange - CTX cyclophosphamide - DC dendritic cell - dUTP deoxyuridine triphosphate - FACS fluorescence-activated cell sorter - FBS fetal bovine serum - FITC fluorescein isothiocyanate - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN- interferon-gamma - IL-4 interleukin-4 - NaS sodium succinate - OCT optimal cutting temperature - PBS phosphate-buffered saline - PI propidium iodide - Tdt terminal deoxynucleotidyl transferase - TNF- tumor necrosis factor alpha - -TOS -tocopheryl succinateSupported by grants 1 RO1 CA94111-02 from the NIH and DAMD 17010126 from the DOD.     

Dendritic cell–tumor cell hybrid vaccination for metastatic cancer

Dendritic cells are the most potent antigen-presenting cells, and the possibility of their use for cancer vaccination has renewed the interest in this therapeutic modality. Nevertheless, the ideal immunization protocol with these cells has not been described yet. In this paper we describe the preliminary results of a protocol using autologous tumor and allogeneic dendritic hybrid cell vaccination every 6 weeks, for metastatic melanoma and renal cell carcinoma (RCC) patients. Thirty-five patients were enrolled between March 2001 and March 2003. Though all patients included presented with large tumor burdens and progressive diseases, 71% of them experienced stability after vaccination, with durations up to 19 months. Among RCC patients 3/22 (14%) presented objective responses. The median time to progression was 4 months for melanoma and 5.7 months for RCC patients; no significant untoward effects were noted. Furthermore, immune function, as evaluated by cutaneous delayed-type hypersensitivity reactions to recall antigens and by peripheral blood proliferative responses to tumor-specific and nonspecific stimuli, presented a clear tendency to recover in vaccinated patients. These data indicate that dendritic cell–tumor cell hybrid vaccination affects the natural history of advanced cancer and provide support for its study in less advanced patients, who should, more likely, benefit even more from this approach.     

Somatodendritic localization of Translin, a component of the Translin/Trax RNA binding complex

Recent studies implicating dendritic protein synthesis in synaptic plasticity have focused attention on identifying components of the molecular machinery involved in processing dendritic RNA. Although Translin was originally identified as a protein capable of binding single-stranded DNA, subsequent studies have demonstrated that it also binds RNA in vitro. Because previous studies indicated that Translin-containing RNA/single-stranded DNA binding complexes are highly enriched in brain, we and others have proposed that it may be involved in dendritic RNA processing. To assess this possibility, we have conducted studies aimed at defining the localization of Translin and its partner protein, Trax, in brain. In situ hybridization studies demonstrated that both Translin and Trax are expressed in neurons with prominent staining apparent in cerebellar Purkinje cells and neuronal layers of the hippocampus. Subcellular fractionation studies demonstrated that both Translin and Trax are highly enriched in the cytoplasmic fraction compared with nuclear extracts. Furthermore, immunohistochemical studies with Translin antibodies revealed prominent staining in Purkinje neuron cell bodies that extends into proximal and distal dendrites. A similar pattern of somatodendritic localization was observed in hippocampal and neocortical pyramidal neurons. These findings demonstrate that Translin is expressed in neuronal dendrites and therefore support the hypothesis that the Translin/Trax complex may be involved in dendritic RNA processing.     

Generation and functional characterisation of dendritic cells from patients with pancreatic carcinoma with special regard to clinical applicability

Background: We studied dendritic cell (DC) function in patients affected by pancreatic carcinoma, and the possibility of obtaining DC adequate for immunological treatment modalities. Methods: Leucocytes were isolated from buffy coats obtained by autotransfusion of six patients undergoing pancreatico-duodenectomy. The leucocytes were cryopreserved and, after thawing, were purified by density gradient and/or plastic adhesion. They were then cultured in vitro in cytokine-enriched medium (granulocyte/macrophage-colony-stimulating factor + interleukin-4) with different sources of serum: 10% fetal calf serum (FCS), 2% autologous human serum or 2% pooled human AB serum. Results: The DC obtained were identical to those from healthy donors in terms of phenotype, antigen uptake capacity, capacity for antigen presentation and their capacity to mature after exposure to stimuli like CD40L. DC differentiated in human serum demonstrated more mature behaviour than did DC cultured in FCS but, after exposure to CD40L, this difference disappeared. In one patient soluble factors in serum were able to inhibit the capacity of DC to stimulate T cells. Conclusion: It's possible to obtain DC from autotransfusion of patients with pancreatic carcinoma: these cells do not show evident quantitative or qualitative alterations, are able to present soluble antigen even when cultured in the presence of human serum and may be used in immunological tumour treatments. Received: 18 May 2000 / Accepted: 27 July 2000