ABSTRACT
BACKGROUND: We hypothesize that dendritic cells (DCs) can process antigens from autologous melanoma apoptotic bodies (MABs) and induce effector T cells in melanoma patients. MATERIALS AND METHODS: Peripheral blood mononuclear cells were obtained from three stage IV melanoma patients and adherent cells were cultured in complete medium (CM) containing GM-CSF (800 U/ml) and IL-4 (1000 U/ml) for 7 days. Autologous MABs from melanoma cells following actinomycin D treatment (0.5 microgram/ml) for 24 hours, were added to 72 hour DC culture. Autologous effector T cells were cultured in CM containing 60 IU/ml of IL-2 and were stimulated by MAB-pulsed DCs three times at a weekly interval. Effector T cells were harvested at the end of third cycle of DC stimulation. RESULTS: Using ELISPOT, IFN-gamma production by effector T cells stimulated by MAB-pulsed DCs was significantly higher than that by T cells without DC stimulation. Microscopy demonstrated phagocytosis of MABs by DCs. CONCLUSIONS: MAB-pulsed DCs are capable of stimulating Th1-directed autologous effector T cells. Pulsing DCs with autologous MABs may be a novel approach in future DC-based immunotherapeutic trials.
Subject(s)
Antigens, Neoplasm/immunology , Dendritic Cells/immunology , Th1 Cells/immunology , Apoptosis/immunology , Dendritic Cells/physiology , Humans , In Vitro Techniques , Interferon-gamma/analysis , Interleukin-10/analysis , Lymphocyte Activation/immunology , Melanoma/pathology , Phagocytosis , Phenotype , T-Lymphocytes/immunologyABSTRACT
Dendritic cells (DCs) are professional antigen presenting cells. Two x 10(7) peripheral blood mononuclear cells (PBMCs) from each of 6 melanoma patients and 4 normal donors were incubated in 6-well plates. The adherent cells were cultured in GM-CSF (800 U/ml) and IL-4 (1000 U/ml) for 10 days. Anti-CD80, anti-CD86, anti-HLA-DR, anti-HLA-class I monoclonal antibodies and supernatant from two melanoma cell lines were incubated with DCs before responder T cells were added in mixed lymphocyte reaction (MLR). The mean yields of DCs were 3.6 x 10(5) and 9.3 x 10(5) for donor and the melanoma patients respectively. MLR was inhibited by anti-CD80, anti-CD86, anti-HLA-DR, anti-HLA-Class I and supernatant of melanoma cultures. The characteristics of DCs generated from melanoma patients were comparable to those from donors. DC function was inhibited by soluble factors in melanoma cultures. Further studies are warranted to characterize these inhibitory factors.
Subject(s)
Dendritic Cells/pathology , Melanoma/blood , Antibodies, Monoclonal/pharmacology , Antigen Presentation/drug effects , Antigens, CD/immunology , B7-1 Antigen/immunology , B7-2 Antigen , Blood Cell Count , Cells, Cultured , Dendritic Cells/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , HLA Antigens/immunology , HLA-DR Antigens/immunology , Humans , Interleukin-4/pharmacology , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Melanoma/immunology , Membrane Glycoproteins/immunology , T-Lymphocytes/immunologyABSTRACT
Enzyme-linked immunospot (ELISPOT) is a sensitive technique for the detection of cytokines released by immune cells. The technique is well established and correlates closely with the enzyme-linked immunosorbent assay (ELISA) technique. Here, we introduce an integrated imaging-analysis system to quantitate the spots formed by the ELISPOT assay. The spots can be easily counted when the background is clear and the spots are few. However, when the number of spots increases to > 100, this task becomes challenging and time-consuming. To minimize time and standardize the counting procedure, we have used a digital camera linked to a computer system for reading the plates. In general, the computer is able to count more spots and has a smaller standard deviation when compared with the manual microscopic count. This integrated system is commercially available, and we believe that this method is objective, time-saving and consistent in routine ELISPOT assay counting, especially when numerous spots are present.
