Phenotypic and functional patterns of murine bone marrow-derived dendritic cells influenced by in vitro differentiation conditions

The selection of an appropriate differentiation protocol for murine bone marrow-derived dendritic cells (BM-DCs) still presents a challenge due to the inherent plasticity of dendritic cells (DCs) and the high diversity of generation methods. As such, it is essential to evaluate in vitro BM-DCs characteristics during initial steps of experimental model development and tailor the protocols according to specific research goals while preserving the functional and phenotypic properties of DCs. In our study, we observed relevant variations in phenotypic and functional characteristics of BM-DCs differentiated with GM-CSF alone (GDCs) compared to those differentiated with GM-CSF and IL-4 (G4DCs). We found that GDCs exhibited high levels of costimulatory molecules, indicating a phenotypically mature state, but preserved the ability to respond to LPS (lipopolysaccharide) maturation stimuli by cytokine production. Moreover, cytokine secretion patterns observed in prime/ boost co-culture systems indicate the potential of Ag-pulsed BM-DCs to prime naive T cells. In contrast, while addition of IL-4 resulted in cells with a more immature phenotype that are responsive to LPS-driven maturation, it also promoted nonspecific T cell signaling that could interfere with in vitro evaluation of priming and antigen-specific T cell responses. These results highlight the impact of specific differentiation conditions that impose on the phenotypic and functional patterns of BM-DCs. Furthermore, the study emphasizes the significance of reassessing, even widely, the already employed protocols for DCs differentiation, and moreover, our study can provide valuable insights and guidance for novel in vitro experimental designs based on BM-DCs.