Introduction: Dendritic cells (DCs) are considered the most powerful antigen-presenting cells (APC) playing a strategic role in triggering the immune response. Monocytes are the physiological precursors of myeloid DCs.
Aims: This paper aims to develop an in vitro experimental model of human dendritic cells generation and phenotypic and functional analysis starting from CD14+ monocyte precursors isolated from peripheral blood.
Materials and methods: CD14+ monocytes were isolated from venous peripheral blood which was harvested from consenting healthy donors. By the means of GM-CSF and IL-4 immature DCs were generated from monocytes. In the sixth day we added TNF-alpha and PGE-2 maturation factors and, after 48 hours we harvested mature DCs. Their morphology was monitored by optical microscopy, while the phenotypes were determined by flow cytometry.
Results: After three days of culture, the monocytes developed a specific morphology, gathering in clusters; cells presenting specific features start to detach in the sixth day. Treatment with TNF-alpha and PGE-2 induces the maturation of DCs and up-regulation of CD80, CD83 and CD86 expressions. Phenotypic analysis of mature DCs was HLA-DR+++/CD86- CD80+++/CD14(-)/CD11a+/CD83+++. DCs obtained are able to stimulate in vivo allolymphocyte capacity. Functional analysis test showed 87% CD4+ CFSE+(DCs+PBMC) cells vs CD4+ CFSE+(PBMC) 15% cells.
Conclusions: This experiment developed an effective and viable way to generate DCs from PBMCs exposed to GM-CSF and IL-4. The cells obtained presented morphological, phenotypic and functional features as DCs described in the literature.