Kinetics of Monocyte Differentiation to Dendritic Cells

Abstract

Dendritic cells (DCs), the professional antigen-sensing and antigen-presentingcomponents of the immune system, play a crucial role in this process by initiating and regulating immune responses. It is well established that the main function of iDC in steady-state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of danger signals and subsequently promote T cell immunity. DCs have been difficult to isolate due to their low concentration in peripheral blood and lack of a DC specific marker,several methods have been developed in order to generate human DCs in vitro. They usually allow one to obtain DCs by the plastic adherence of CD14‏ monocytes from PBMC, followed by 5 to7 days incubation in GM-CSF and IL-4 containing medium. The ready culture systems not only permit detailed studies of DC biology, but also hold promises for DC to become widespread used in medical practice such as immunotherapy in cancer, some infectious diseasesand autoimmune diseases. In this study, different trials were done to study kinetics of monocyte differentiation to DCs after treatment with specific growth factors.Fresh citrated blood samples were taken from healthy blood donors. PBMCs were isolated from freshly collected buffy coats by density gradient centrifugation. CD14+ monocytes were purified by MACS. CD14+wereabout 75-85% by flow-cytometry.Cells were cultured in complete RPMI media supplemented with differnet concentrations of IL-4 and GM-CSF. In this study it was better to use growth factor in a conc. 0.5μl/ml and IL-4 in a conc. 1μl/ml from day 0 in culture media for dendritic cell generation. Harvest cells on 5th day for their better viability than 7th day. Using marker combination CD14–HLA-DR+ CD11c+ to detect dendritic cell generation.Successful differentiation into immature MoDCs being CD14-CD11c+HLA-DR+by the 5th day was confirmed by flow-cytometry. These cells could be promising in clinical settings through synthesis of DC vaccines for the treatment of autoimmune and allergic diseases as well as in prevention of graft rejection being tolerogenic in nature. Also they may undergo maturation using certain cytokines as TNF-α or by using LPS to be able to stimulate the immune response against various infections as well as tumor cells. In conclusion, whatever cell source is chosen to generate a specified cellular product for treatment of human diseases, it should comply with important clinical requirements including patient safety and source availability.