Competent antigen-presenting cells are generated from the long-term culture of splenocytes with granulocyte-macrophage colony-stimulating factor

•Long-term culture of mouse splenocytes with GM-CSF produces CD11b⁺CD11c⁺ cells.•Growth of splenocytes in the culture with GM-CSF is independent of FLT3/FLT3L signaling.•CD11b⁺CD11c⁺MHC IIhi cells from the culture exhibit authentic morphology and function of dendritic cells.

Dendritic cells (DCs) are routinely produced from the culture of mouse bone marrow (BM) with granulocyte-macrophage colony-stimulating factor (GM-CSF) within a period of 10 days. Although splenic extramedullary myelopoiesis was suggested to occur under the influence of GM-CSF, the hematopoietic outcome of splenic culture with GM-CSF has not been scrutinized. We have cultured mouse splenocytes with GM-CSF for an extended period of time, where we discovered that the CD11b⁺CD11c⁺ cells began to proliferate prominently after 10 days and their number increased until the 4th week of the culture. In parallel experiments, FMS-like tyrosine kinase 3 (FLT3) and its ligand, FLT3L, were not found to influence the culture of splenocytes. Like DCs in the culture of BM with GM-CSF, a distinct population of CD11b⁺CD11c⁺MHC IIhi cells was readily identified as DCs in the long-term culture of splenocytes. After being isolated and plated overnight the CD11b⁺CD11c⁺MHC IIhi cells exhibited non-adherent dendritic morphology, while the other CD11b⁺CD11c⁺ cells became adherent. Besides, these CD11b⁺CD11c⁺MHC IIhi cells possessed relatively weak endocytic and phagocytic abilities but displayed strong antigen-presenting capacities, revealing DC-like characteristics; in contrast, the other CD11b⁺CD11c⁺ cells showed strong endocytosis and phagocytosis of antigens but were poor at antigen presentation, indicating macrophage-like traits. Therefore, we demonstrated that phenotypically as well as functionally genuine DCs are generated in the long-term culture of splenocytes with GM-CSF.