Abstract
Long-lived plasma cells (PCs) located within the bone marrow are the source of protective antibody for extended periods of time. Recent studies pointed out that PC survival depends on the ability of PCs to find a suitable niche. Bone marrow environment of PCs is constituted by cells of either mesenchymal (stromal cells and osteoblasts) or hematopoietic origin (macrophages, dendritic cells, osteoclasts (OCs)). In this work, we evaluated the ability of these five types of cells to support the long-term survival of human PCs in vitro.
Stromal cells were derived from normal bone marrow mononuclear cells and osteoblasts were SAOS2 cells (malignant osteoblastic cells). Macrophages, dendritic cells and OCs were derived from adherent circulating mononuclear cells with M-CSF, IL4 and GM-CSF or RANKL and M-CSF, respectively. Morphology, phenotype and functional features were used to characterize each cell type. On the other hand, plasmablasts (PBs) were generated from tonsil CD27+ B cells activated by CD40L expressing fibroblasts and then induced to differentiate by removal of fibroblasts in the continuous presence of IL2 and IL10. PBs were purified (by CD20 depletion) and cocultured with the five types of cells previously described.
To assess PC differentiation and survival in cocultures, we monitored the frequency of PCs by flow cytometry using an anti-CD38 and anti-CD138 double staining, along with Ig accumulation. Among cells of either mesenchymal (stromal cells and osteoblasts) or hematopoietic origin (macrophages, dendritic cells, OCs), only OCs allowed PBs to survive and differentiate into PCs that remained alive more than 14 days. Indeed, after a 5-day coculture with OCs, 100 000 PCs were alive (400 000 PBs were seeded at day 0) as compared to less than 10 000 PCs in the other culture conditions. Moreover, the total frequency of PCs after 14 days in culture with OCs was approximately 5% of the original input at day 0. PCs fully maintained their antibody secretory capacity since the level of secreted antibodies increased. Cellular interactions were required since PBs cultured with either OC conditioned medium or with OCs in transwell did not survive nor differentiate. Identification of membrane and soluble molecules supporting PC survival is now under investigation.
Our data indicate that the interaction with OCs is not limited to malignant PCs i.e., myeloma cells, and pointed out that OC is an essential partner of normal PC.
Adult Bone Marrow Stromal Cells in the Embryonic Brain: Engraftment, Migration, Differentiation, and Long-Term Survival
