The effect of melanoma cell coculture on generating human dendritic cells(DC) from CD14+ monocytes has not been extensively studies yet. We investigated how melanoma cells and membrane-bound granulocyte/macrophage colony stimulating factor (mbGM-CSF) melanoma cell lines affect the differentiation of DC obtained by in vitro coculture of plastic-adherent blood monocytes with the presence or absence of GM-CSF and interleukin(IL)-4.

The malignant melanoma cell lines(Conley and Jorp) were used in this study. Conley B-F8 and Jorp C-E6 are mbGM-CSF-positive cells derived from Conley and Jorp respectively. Each cell-free supernatants were collected and used to assess the level of GM-CSF using ELISA. Peripheral blood was collected from healthy donors. Adherent monocytes were cocultured for 6-7 days with irradiated mbGM-CSF and wild type melanoma cells(50 Gy) at each 1:1 and 0.1:1 ratio in 6-well culture plates in X-vivo culture medium containing IL-4. In control experiments, GM-CSF was added at the time of culture initiation. Immunophenotyping was performed by triple color immunofluorescence staining with flow cytometry analysis.

In this study, GM-CSF was detected at low levels in the culture supernatants of Conley B-F8 (0.48 ng/106 cells/24h), whereas there was no detectable GM-CSF in that of Conley melanoma cell line. ELISA demonstrated that Jorp C-E6 cells produced 18 ng/106 cells/24h, whereas Jorp wild type produced 3.6 pg/106 cells/24h. Monocytes cultured with GM-CSF/IL-4 generated the expression of high levels of HLA DR, CD1a and CD86, while the expression of CD14 and CD83 were in low amounts. After culture in medium containing IL-4 alone, monocytes cocultured with Conley B-F8 showed low levels of CD1a and CD86 and relatively low value of HLA DR. However, the addition of GM-CSF to these cultures resulted in an increased expression of these markers and decreased that of CD14. Monocytes cocultured with Jorp C-E6 illustrated similar expression pattern of CD1a, HLA DR and CD14 in the presence or absence of GM-CSF as Conley B-F8 melanoma cell line. Monocytes cocultured with Conley B-F8 melanoma cells at 1:1 and 0.1:1 ratio showed no significant difference in expression of CD1a, CD14 and CD83 between two ratio.

Our results illustrate the feasibility to generate monocyte-derived DC from coculture with melanoma tumor cells in the presence of GM-CSF and IL-4. However, mbGM-CSF tumor cells did not significantly enhance the DC differentiation through juxtacrine stimulation unless soluble GM-CSF was added in the culture media.