?(Fig

?(Fig.2a).2a). 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and Nafamostat increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas. and a melanoma cell lysate, referred to as TRIMEL, showed effectiveness in improving long-term survival in vaccinated patients with advanced malignant melanoma (MM).9,21 Moreover, it was demonstrated that TRIMEL by itself can rapidly induce a mature and committed DC phenotype from activated monocytes (AMs), even in the absence of pro-inflammatory cytokines.22,23 Furthermore, the presence of damage-associated molecular patterns, as derived from stressing the human metastatic melanoma cell lines constituting TRIMEL with heat-shock, is responsible for an efficient antigen cross-presentation by TAPCells.23 However, the migration ability of TAPCells to draining lymph nodes, a relevant prerequisite for its clinical efficacy, remains to be studied. To investigate whether patient-derived TAPCells are able to migrate to draining lymph nodes in an system, we established a xenograft ectopic animal model using immunodeficient or natural killer (NK) -depleted immunocompetent mice. We also tested if TRIMEL was involved in the increased expression of surface CCR7 receptors during the differentiation and maturation of TAPCells from the monocytes of MM patients. Furthermore, it was important to test the lysate effect in a stable cell line model, such as the monocyte/macrophage THP-1, because monocytes derived from patients Nafamostat can show genotypic variations that could eventually affect the clinical outcome of treated patients.24 Using assays, we showed that TAPCells and TRIMEL-stimulated THP-1 cells were capable of specifically migrating in the presence of the canonical CCR7 ligand, CCL19. Finally, we demonstrated by flow cytometry and immunohistochemistry that TAPCells are able to migrate from the injection site into draining lymph nodes. This work contributes to a further understanding of the effect of tumour cell lysates on APCs generated and helps in the design of new effective strategies for DC-based vaccine therapies for MMs. Materials and methods PatientsPeripheral blood mononuclear cells were obtained by leukapheresis from four advanced (stage IV) MM patients (codes MT-123, MT-197, MT-198 and MT-199), who were treated using a previously reported autologous TAPCell vaccination protocol.23,21 Part of the peripheral blood mononuclear cells was then used for TAPCell generation for and assays. The present study was performed in agreement with the Helsinki Declaration and approved by the Bioethical Committee for Human Research of the Faculty of Medicine, University of Chile. All patients signed informed consent forms for the planned experiments. Mice strainsSix- to 8-week-old male C57BL/6J (C57) and NOD.Cg-(US Biological) or with only the medium. Flow cytometryTAPCells were characterized phenotypically by flow cytometry using the L1CAM following conjugated antibodies: anti-HLA-DR-FITC, CD80-FITC, CD83-FITC, CD86-FITC, CD11c-PE-Cy7 and CCR7-FITC (eBioscience, San Diego, CA). Briefly, cells were gently removed from the culture plates using a cell scraper. Then, the cells were centrifuged at 193 for 5 min at 4, washed with PBS and incubated with antibodies for 30 min. After being washed twice with PBS, samples were acquired on a FACSCalibur (BD Biosciences) and analysed using FlowJo software (Tree Star, Inc., OR). Cell viability was verified through trypan Nafamostat blue exclusion, and over 95% of treated cells in all cases excluded trypan blue. All the analyses were made in the Compact disc11c+ cell people of every test and state. To judge DC migration by FACS evaluation, TAPCells Nafamostat and AMs had been labelled using the fluorescent dye PKH67 (Sigma-Aldrich, St Louis, MO). Quickly, 18 106 cells in 18 ml diluent C had been blended with 27 l PKH67 dissolved in 3-ml diluent C and stained for 5 min at area heat range. Labelling was ended by incubation with 24 ml of 100% FBS. From then on, cells were cleaned double in RPMI supplemented with 10% FBS. Real-time quantitative.