Culture of Dendritic Cell from Normal Peripheral Blood Monocyte and Its Anti-tumor Immune Activity When Pulsed by Renal Cell Carcinoma Cell Line: In vitro Study

Cheon, Sang Hyeon; Chung, Han; Shin, Yoon Joo; Kim, Choung Soo

Abstract

PURPOSE: Dendritic cells (DC) play a crucial role in the initiation of primary immune response and are known as an excellent adjuvant for anti-cancer immunity. In this study, we tried to obtain substantial numbers of DC from peripheral blood of normal volunteers. We also investigated the anti-tumor immune response of DC pulsed by renal cell carcinoma cell line A498 in vitro.
MATERIALS AND METHODS
DC were generated by culturing plastic adherent mononuclear cells from the peripheral blood in the presence of granulocyte-macrophage colony- stimulating factor and interleukin-4. Immature DC were cocultured with T-cells and pulsed by A498. MTT analysis was performed using the medium in which A498 only was cultured as control. Our experiments were analyzed by means of a commercial IL-12 p70 ELISA (Quantikine; R & D Systems, Minneapolis, MN). The capture antibodies used in both tests specifically recognize the p70 heterodimer, but not the free p40 chains. Detection limits were 30pg/ml of IL-12.
RESULTS
We could obtain 1.5-2.0x106 DC with phenotype typical of mature DC (CD14-, CD80+, and CD83+) from the normal peripheral blood. On T-cell proliferation assay, the number of T-cells increased in proportion to that of DC and when DC were pulsed by A498, the same phenomenon could be observed. DC and T-cell media with A498 tumor lysate showed more production of IL-12 on IL-12 p70 ELISA than the media without A498 tumor lysate.
CONCLUSIONS
We could successfully obtain mature DC from the peripheral blood. The data revealed indirectly that DC treated with tumor lysate enhance immune activity and thereby increase the anti-cancer effect of T-cells. Further investigations including in-vivo study are necessary to realize the effect of immunotherapy using DC against metastatic renal cell carcinoma.